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International Journal of Phytomedicine and Phytotherapy

Clinical Phytoscience Cover Image
  • Original contribution
  • Open Access

Antioxidant, antimicrobial, and GC-MS profiling of Saussurea obvallata (Brahma Kamal) from Uttarakhand Himalaya

Clinical PhytoscienceInternational Journal of Phytomedicine and Phytotherapy20195:12

https://doi.org/10.1186/s40816-019-0105-3

  • Received: 26 June 2018
  • Accepted: 21 January 2019
  • Published:

Abstract

Background

Saussurea obvallata (DC.) Edgew. (S. obvallata) is an endangered medicinal herb of the high altitude Himalayan region with immense cultural significance. This study is one of the first report of pharmacological evaluation and GC-MS analysis of methanolic extracts of leaves and flowers of S. obvallata.

Methods

Total flavonoid content (TFC) and total phenolic content (TPC) were performed according to standard protocols. The antioxidant activities of both the extracts (methanol and aqueous) were examined using two complementary methods, namely diphenylpicrylhydrazyl (DPPH) and Hydrogen peroxide (H2O2) assay. While, antimicrobial potential examined by using classical assays on pathogenic microbial strains along with appropriate controls of antimicrobial agents. Gas Chromatographic-Mass Spectrometric analyses of methanolic extracts of S. obvallata were performed for the identification of bioactive components. Statistical analysis was performed by using SPSS version 16.0.

Results

The extracts of leaves and flowers of S. obvallata showed significant results for all pharmacological experiments. TPC was recorded from a range of 132.67 ± 0.17 to 358.67 ± 0.17 mg GAE gm− 1 dw while TFC was recorded from a range of 65.73 ± 1.55 to 326.88 ± 2.11 mg QE gm− 1 dw. Per cent DPPH free radical scavenging activity was recorded from a range of 29.25 ± 0.86 to 82.88 ± 0.48% whereas per cent H2O2 free radical scavenging activity were recorded from a range of 39.75 ± 0.36 to 41.05 ± 0.46%. Antimicrobial activity was analyzed against four bacterial strains and three fungal strains. S. obvallata extracts showed fine zone of inhibition against Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus, but Escherichia coli showed resistant (in terms of lower zone) against the extract compared to three bacteria. In antifungal activity, extract showed maximum zone of inhibition against Candida glabrata followed by Candida albicans and Candida tropicalis. GC-MS analysis of methanolic extracts of leaves and flowers of S. obvallata showed the presence of 36 and 48 components, respectively, based on retention time (Rt), and area per cent.

Conclusion

These findings confirm the traditional claims and contribute in providing promising baseline information for the pharmacological use of S. obvallata. Additional highly developed research is essential for isolation and identification of specific active components which are responsible for pharmacological properties of the plant.

Keywords

  • Antioxidant
  • Antimicrobial
  • Microbial strains
  • Pharmacological evaluation
  • Saussurea obvallata
  • Traditional claims

Background

Herbal drugs are used in the traditional Indian system of medicine and also in other systems of medicine in the world for times immemorial due to low cost, effectiveness and no or low side effects. These attributes of the so called “natural” medicines are attracting the attention of increasingly large number of people of the world. World Health Organization (WHO) survey reports that 80% of the population in developing countries are still dependent on the traditional and folk systems of medicine, 85% traditional medicines are prepared by the use of plant extracts and that nearly 70% of prescribed human medicines are derived from the plants [13]. International market of herbal drugs is around US $ 62 billion, which is poised to grow to US $ 5 trillion by 2050 in the Asian and International market [4]. Demand of herbal drugs in India alone is estimated at US $ 1 billion per year [5], and Uttarakhand is one of the richest source for a whole variety of medicinal plants.

Saussurea is a very important genus of the family Asteraceae due to a variety of reasons, and most species of this genus are well investigated at the international level [69]. However, little is known about S. obvallata (2n = 32). It is an endemic herb of the Himalayan region and distributed between 3000 and 4800 m amsl. Commonly it is known as Brahma Kamal and is the state flower of Uttarakhand (India). It is a well known plant across Uttarakhand due to traditional, medicinal, ornamental and religious purposes [10]. It is used for the treatment of various diseases or disorders like paralysis, cerebral ischemia, wounds, cardiac disorders and mental disorders; some people also use it as antiseptic, in healing cuts, etc. [10]. Preliminary investigations about the phytochemicals found in S. obvallata (qualitative) have been reported by Semwal et al. [11], and its mineral composition has been described by Mishra et al. [12]. The present study, deals with the estimation of its total phenolic and flavonoid contents, as well as antioxidant and antimicrobial activities associated with its extracts. Gas Chromatography-Mass Spectrometry (GC-MS) based analyses of crude methanolic extracts of S. obvallata leaves and flowers have been carried out for the identification of active components.

Methods

Collection of plant material

The plant material (leaves and flowers) was collected from Kedarnath valley (4335 m amsl, 30°40′73” N latitude and 79°06′20″ E longitude) in Uttarakhand, India, during the month of September, 2012. The identity was subsequently confirmed by Dr. Anup Chandra (Scientist–E) Systemic Botany, Forest Research Institute, Dehradun, Uttarakhand, India (Ref. n. GEU/DBT/AT-1PS/2013). The fresh material was kept in perforated poly bags and immediately brought to the laboratory.

Preparation of extracts

The extracts were prepared as per Sati and Joshi [13] with slight modification. The flower and leaf samples of S. obvallata were washed, dried, and powdered using an electric blender (Willey Grinder Mill, Micro Scientific, India). Two solvents, methanol or water, were used for the extraction of phyto-constituents from the dried powder.

Methanol

Methanolic extracts of leaves and flowers were prepared by the Soxhlet extraction procedure. In this method the powdered plant material (1.0 g) of leaves and flowers were extracted in 10 mL of 70.0% (v/v) methanol used and extraction was carried out for 5 h. Afterwards the extract was collected and filtered through two layers of muslin cloth and then the filtrate was transferred to a rotator vacuum evaporator (Strike-12, Steroglass, Italy) flask and dried under reduced pressure and temperature less than (50 °C). Then dried extract was stored at (− 20 °C) until used. The percentage yield of the plant extract was calculated according to the formula.
$$ \left[\mathrm{Percent}\ \mathrm{Yield}\ \mathrm{of}\ \mathrm{the}\ \mathrm{extract}\ \left(\%\right)={\mathrm{C}}_{\mathrm{X}}/{\mathrm{C}}_{\mathrm{Y}}\times 100\right] $$
(1)

Where, CX = Plant material weight after extraction process, CY = Plant material weight taken for extraction.

Water

Aqueous extracts of leaves and flowers were prepared using maceration technique. In this method the powdered plant material (1.0 g) of leaves and flowers were extracted in 10 mL of 100% (v/v) distilled water and allowed it to stand for 48 h (Shaker, 25 ± 1 °C). Afterwards the extract was filtered through two layers of muslin cloth and transferred to a rotator vacuum evaporator (Strike-12, Steroglass, Italy) flask and dried under reduced pressure and below 50 °C. The dried extract was stored at − 20 °C until used. The per cent yield of the extract was calculated using the formula giving in methanolic extraction.

Determination of total phenolics

The total phenolic content (TPC) in methanolic and aqueous extracts was determined by the Folin-Ciocalteu’s reagent colorimetric method described by Anand et al. [14]. Both the methanolic and aqueous extracts of S. obvallata (20 μL) were taken from the stock solutions (1 mg mL− 1) of methanol and aqueous solutions, diluted with distilled water (80 μL); this was then mixed with Folin-Ciocalteu reagent (500 μL) and allowed to react for 5 min in the dark (25 ± 1 °C). The mixture was neutralized by adding 400 μL of 7.5% sodium carbonate and kept in the dark (25 ± 1 °C, 30 min). The absorbance of this blue colored mixture was measured at 765 nm using a spectrophotometer (Thermo Scientific UV- 10). Quantification was done on the basis of a standard curve prepared using gallic acid (Sigma Aldrich, G7384-100G) at the concentration of 0.2, 0.4, 0.6, 0.8, and 1.0 mg mL− 1 in 70.0% methanol. Based on the absorbance values, the TPC was estimated (mg mL− 1) from the calibration curve and the results expressed in terms of mg gallic acid equivalent dry weight (GAE g− 1 dw). The whole process of TPC estimation was done and experiment was performed in triplicate.

Determination of total flavonoids

The total flavonoid content (TFC) was determined using the aluminium chloride colorimetric method, as described by Anand et al. [14]. Briefly, the methanolic or aqueous extracts of S. obvallata (100 μL) from the respective stocks (1 mg mL− 1) of methanol or aqueous solutions were first diluted with distilled water (400 μL) and then 30 μL of 5.0% aq. NaNO2 (sodium nitrite) solution was added to them. After 5 min, 30 μl of 10.0% aq. AlCl3 (aluminium chloride) was added, and the mixture was allowed to stand for 5 min (25 ± 1 °C). Subsequently 20 μL of 4.0% aq. NaOH (sodium hydroxide) solution was added and the volume was made up to 1.0 mL with distilled water (420 μL). The absorbance of this mixture was immediately measured using a UV spectrophotometer (Thermo Scientific UV- 10) at 510 nm. Quantification was carried out on the basis of a standard curve prepared with quercetin (Sigma Aldrich, 337,951–25G) at the concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg mL− 1 in 70.0% methanol. The results were expressed in terms of mg quercetin equivalent dry weight (QE g− 1 dw). The whole process of TFC estimation was carried out in triplicate.

Determination of antioxidant activity by 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) method

The free radical scavenging activity of samples was assessed according to the method of Azzahra et al. [15]. Briefly, first the stock solution of DPPH (Merck-Millipore, 300,267-50MG) was prepared by dissolving 24 mg DPPH in 100 mL methanol and the solution was stored at − 20 °C until used. Subsequently, the working solution was prepared by mixing 10 mL of stock with 60 mL of methanol, which gave an absorbance of 1.10 ± 0.02 units at 515 nm. Both the extracts (10 μL) were mixed with DPPH (990 μL) and the reaction was allowed to proceed for 2 h (25 ± 1 °C) in the dark, and then the absorbance was recorded at 515 nm. Ascorbic acid (Sigma Aldrich, A92902-25G) was used as a standard for making the calibration curve using 0.2, 0.4, 0.6, 0.8, and 1.0 mg mL− 1 concentrations of the standard. The estimations were carried out in triplicate, and the per cent scavenging activity was calculated using the formula:
$$ \left[\mathrm{Scavenging}\ \mathrm{rate}\ \left(\%\right)=\left[\left(\mathrm{Ac}-\mathrm{Ae}\right)/\mathrm{Ac}\right]\times 100\right] $$
(2)

Where, Ac and Ae represent the absorbance values of the control and extract, respectively.

Determination of antioxidant activity by hydrogen peroxide (H2O2) method

Determination of antioxidant activity by this method was carried out according to Panovska et al. [16]. Several oxidases generate H2O2, which is scavenged, either directly or indirectly via its reduction product, hydroxyl radical. In this method, when a scavenger is incubated with hydrogen peroxide, the decomposition or loss of H2O2 is measured using UV Spectrophotometer. Extracts (1 mL) were mixed with 20 mM H2O2 solution (2 mL) in phosphate buffer saline (PBS solution pH 7.5). The absorbance was measured after 10 min at 230 nm against phosphate buffer. H2O2 solution (2 mL) and the solvent (1 mL) were used as control and PBS as blank. Ascorbic acid (Sigma Aldrich, A92902-25G) was used as standard and the calibration curve was prepared using 0.2, 0.4, 0.6, 0.8, and 1.0 mg mL− 1 concentrations of the standard. The per cent scavenging rate was calculated using the formula given for DPPH method.

Determination of antibacterial activity

The antimicrobial activity of S. obvallata extracts was carried out using standard protocols. Bacterial and fungal strains were obtained from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. Selection of bacterial and fungal strains was made on the basis of their pathogenic nature, wider availability, antibiotic resistance and popularity. The bacterial strains included three Gram-negative strains namely, Pseudomonas aeruginosa (MTCC 4306), Escherichia coli (MTCC 1698), and Klebsiella pneumoniae (MTCC 9544) and one Gram-positive strain namely, Staphylococcus aureus (MTCC 6908). The antibacterial activity of the extracts was estimated using the well diffusion method [17, 18]. Briefly, the extracts (methanolic or aqueous) were tested on Luria Bertani agar (LB agar; Himedia, Delhi, India) plates to detect antibacterial activity, if any, against P. aeruginosa, E. coli, K. pneumoniae and S. aureus. Bacterial strains were cultured in LB broth at 37 °C for 18 h. The cultures were diluted to 1 × 106 cells mL− 1 in LB broth and 100 μL of each was spreaded onto a separate LB agar plate. Wells of 6 mm diameter were bored using sterile cork borer. The extract (20 μL) from individual stocks (5 mg mL− 1 concentration), and ampicillin (20 μL) from stock (1 mg mL− 1 concentration) were poured in the wells and the plates were further incubated at 37 °C for 24 h. Ampicillin was used as a positive control, whereas DMSO (5%) was used as a negative control. These tests were repeated three times to estimate consistency. Inhibition was measured in terms of zone of inhibition (ZOI) in mm formed on the plates.

Determination of antifungal activity

The well diffusion method was also used to screen the antifungal activity of S. obvallata extracts against three fungal strains of Candida namely, C. albicans (MTCC 3017), C. glabrata (MTCC 3019) and C. tropicalis (MTCC 3416) according to the same protocol [17, 18] giving in the previous section (antibacterial activity). These strains were also procured from the Institute of Microbial Technology (IMTECH), Chandigarh, India. Potato dextrose agar (PDA; Himedia, Delhi, India) was used as the culture medium. Fluconazole (1 mg mL− 1) was used as a control drug (positive) and 5% DMSO was used as a negative control. The extract (20 μL) from individual stocks (5 mg mL− 1) was placed in each well, and the inoculated plates were incubated at 28 °C for 15 h. These tests were done in triplicate and the average of three separate measurements was taken for the colony diameter (mm) of the fungus.

Gas chromatography-mass spectrometry (GC–MS) analyses

GC-MS analyses and component identification of methanolic extracts of flowers and leaves of Saussurea obvallata were performed at the University Science Instrumentation Centre, Jawaharlal Nehru University, Delhi, India. The analyses were performed according to Ezhilan and Neelamegam [19], and Das et al. [20]. The GC-MS system (Shimadzu QP2010PLUS, Kyoto, Japan) was used for the analyses, which was equipped with an auto injector (AOC-20i), head space sampler (AOC-20s), and a fused silica capillary column Rtx-5 (30 m in length × 0.25 mm internal diameter × 0.25 μm film thickness). The oven temperature was set at 100 °C for 2 min, then increased to 250 °C at a rate of 5 °C per min, and finally to 280 °C at a rate of 10 °C per min. One μL of each sample (1 mg mL− 1 stock) was injected to the column in split mode (split ratio 10) with helium (99.99% purity) as a carrier gas with a flow rate of 1.21 mL per min. The presence of unique peak fragmentation patterns for various metabolites/ phytocomponents was detected by an MS detector in full scan mode. Identification of components was based on their retention time (RT) and mass measured under identical GC-MS conditions. Quantitative determinations in the different samples were carried out using the peak areas. Identification of phytocomponents of Saussurea obvallata extracts was confirmed by comparing the spectral data of peaks with the standard mass spectra from the library data base [National Institute of Standards and Technology library (NIST08) and WILEY8]. Total running time of GC-MS was 50.74 min and the relative percentage of individual constituents was expressed on the basis of peak area normalization.

Statistical analysis

The results of phytochemical analyses and antimicrobial activity data were subjected to analysis of variance using SPSS version 16.0. The significance level was determined at p < 0.05 and the means were separated using Duncan’s multiple range test (DMRT), to determine if the values were significantly different. Data have been presented as mean values ± standard error (SE).

Results and discussions

Total phenolic and flavonoid content

Significant variation (p < 0.05) was recorded in the total content of phenolics (TPC) and flavonoids (TFC) in S. obvallata, and the values for TPC and TFC were expressed in terms of mg gallic acid equivalent g− 1 dw (the standard curve equation: y = 1.992x ˗ 0.226, R2 = 0.977) and quercetin equivalent g− 1 dw (the standard curve equation: y = 0.284x + 0.116, R2 = 0.975), respectively. The maximum and minimum TPC values were found in methanolic leaf extract and aqueous flower extract of S. obvallata (358.66 ± 0.17 and 132.67 ± 0.17 mg GAE g− 1 dw), respectively. Intermediate values were recorded for aqueous leaf extract (139.50 ± 0.28) and methanolic flower extract (185.00 ± 0.00). The maximum and minimum TFC values were recorded in aqueous flower extract and methanolic flower extract of S. obvallata (326.88 ± 2.12 and 65.73 ± 1.55 mg QE g− 1 dw), respectively. Intermediate values were recorded for aqueous leaf extract (229.46 ± 1.54) and methanolic leaves extract (188.38 ± 1.01). The results of TPC and TFC estimations have been represented in Table 1. These types of poly phenolic compounds are known to show different types of biological effects, e.g., antioxidant and antimicrobial activities, etc. [21].
Table 1

Quantitative estimation of total phenolic, total flavonoid and free radical scavenging activity by DPPH and H2O2 assays in aqueous and methanolic extracts of leaves and flowers of Saussurea obvallata

Plant extract

Yield of the extracts (%)

TPC (mg GAE g−1 dw ± SE)

TFC (mg QE g− 1 dw ± SE)

DPPH (% S. activity ± SE)

H2O2 (% S. activity ± SE)

L1

13.70

139.50 ± 0.29c

229.46 ± 1.55b

49.67 ± 0.38c

39.75 ± 0.36

L2

19.80

358.67 ± 0.17a

188.38 ± 1.02c

73.48 ± 0.44b

41.05 ± 0.46

F1

13.14

132.67 ± 0.17d

326.88 ± 2.11a

29.25 ± 0.86d

40.12 ± 0.40

F2

11.67

185.00 ± 0.00b

65.73 ± 1.55d

82.88 ± 0.48a

40.76 ± 0.27

Values are expressed in mean ± standard error; Mean values followed by the same letter (s) in a column are not significantly different (p < 0.05) based on DMRT; TPC total phenolic content, TFC total flavonoid content, DPPH 1, 1-Diphenyl-2-picrylhydrazyl, H2O2 hydrogen peroxide, L1& L2 aqueous and methanolic extract of leaves, F1 & F2 aqueous and methanolic extract of flowers, respectively

DPPH and H2O2 free radical scavenging activity

Antioxidant activity was measured by two in-vitro assays and significant variation (p < 0.05) was recorded in antioxidant activity of S. obvallata extracts using DPPH assay, while significant variation (p < 0.05) was not recorded in H2O2 assay. The maximum and minimum per cent DPPH free radical scavenging activity were recorded in the methanolic and aqueous extracts of flowers (82.88 ± 0.48% and 29.25 ± 0.86%), respectively. Intermediate values of DPPH free radical scavenging activity were recorded for aqueous extract of leaves (49.66 ± 0.38%) and methanolic extract of leaves (73.48 ± 0.44%). On the other hand, the maximum and minimum per cent H2O2 free radical scavenging activity were recorded in methanolic leaf extract and aqueous leaf extract (41.05 ± 0.76% and 39.75 ± 0.36%), respectively. Intermediate values of H2O2 free radical scavenging activity were recorded for aqueous extract of flowers and methanolic extract of flowers (40.11 ± 1.10% and 40.76 ± 0.26%).

Antibacterial activity

Antibacterial activity of the extracts of leaves and flowers of Saussurea obvallata was measured in terms of zone of inhibition (ZOI) against Pseudomonas aeruginosa (PA), Escherichia coli (EC), Staphylococcus aureus (SA), and Klebsiella pneumoniae (KP). The maximum zone of inhibition (ZOI) for the aqueous extract of leaves was 20.43 ± 0.30 mm for SA and minimum ZOI was 11.57 ± 0.23 mm for EC. Intermediate ZOI (16.60 ± 0.21, and 14.90 ± 0.21 mm) values were recorded for PA and KP, respectively. The maximum ZOI for methanolic extract of leaves was 19.90 ± 0.56 mm for KP and minimum ZOI was 10.83 ± 0.43 mm for EC, while intermediate values for ZOI (13.90 ± 0.21 mm and 13.60 ± 0.21 mm) were recorded for PA and SA, respectively. The maximum ZOI for the aqueous extract of the flowers was 20.50 ± 0.26 mm for PA and minimum value for ZOI was 13.90 ± 0.21 mm for EC, while intermediate values for ZOI of 16.53 ± 0.24 mm and 18.53 ± 0.24 mm were recorded for SA and KP, respectively. The maximum ZOI for methanolic extract of flowers of S. obvallata was found to be 16.33 ± 0.17 mm for PA and minimum ZOI value of 8.86 ± 0.18 mm was observed for EC, while 12.83 ± 0.16 mm and 11.53 ± 0.24 mm values for ZOI were recorded for SA and KP, respectively. Ampicillin (positive control) showed maximum ZOI against PA (34.33 ± 0.33 mm) followed by SA (31.33 ± 0.33 mm), EC (25.66 ± 0.33 mm), and KP (24.6 ± 0.30 mm). Five per cent DMSO (negative control) did not show any ZOI for individual bacterial strains. In conclusion, it was observed that the extract of S. obvallata caused lower values for ZOI against EC in comparison to other bacteria (SA, PA & KP). The results of antibacterial activity of extracts of S. obvallata against four bacterial strains have been represented in Table 2.
Table 2

The antibacterial and antifungal activity of the extracts of leaves and flowers of Saussurea obvallata determined using the well diffusion method

Plant Extracts

Assay for antibacterial activity

Assay for antifungal activity

P. aeruginosa

E. coli

S. aureus

K. pneumoniae

C. glabrata

C. albicans

C. tropicalis

L1

16.60 ± 0.21c

11.57 ± 0.23c

20.43 ± 0.30b

14.90 ± 0.21d

13.90 ± 0.17b

15.43 ± 0.30bc

8.30 ± 0.15d

L2

13.90 ± 0.21e

10.83 ± 0.43c

13.60 ± 0.21d

19.90 ± 0.56b

14.57 ± 0.23b

15.90 ± 0.56b

13.17 ± 0.09b

F1

20.50 ± 0.26b

13.90 ± 0.21b

16.53 ± 0.24c

18.53 ± 0.24c

14.30 ± 0.12b

14.27 ± 0.50c

12.33 ± 0.18b

F2

16.33 ± 0.17d

8.87 ± 0.19d

12.83 ± 0.17e

11.53 ± 0.24e

14.30 ± 0.47b

15.27 ± 0.47bc

10.90 ± 0.46c

Drug

34.33 ± 0.33a

25.67 ± 0.33a

31.33 ± 0.33a

24.60 ± 0.31a

22.40 ± 0.31a

25.93 ± 0.54a

25.97 ± 0.49a

DMSO

00.00 ± 0.00

00.00 ± 0.00

00.00 ± 0.00

00.00 ± 0.00

00.00 ± 0.00

00.00 ± 0.00

00.00 ± 0.00

Mean values ± SE followed by the same letter (s) in a column are not significantly different (p < 0.05) based on DMRT; P. aeruginosa: Pseudomonas aeruginosa; E. coli: Escherichia coli; S. aureus: Staphylococcus aureus; K. pneumoniae: Klebsiella pneumoniae; C. albicans: Candida albicans; C. glabrata: Candida glabrata; C. tropicalis: Candida tropicalis. L1& L2: aqueous and methanolic extract of leaves, respectively. F1 & F2: aqueous and methanolic extract of flowers, respectively. Ampicillin and fluconazole were used as positive controls for antibacterial and antifungal assays, respectively. DMSO used as a negative control

Antifungal activity

The antifungal activity of extracts of leaves and flower of S. obvallata were assessed against three fungal species, Candida albicans (CA), Candida glabrata (CG) and Candida tropicalis (CT) in terms of zone of inhibition (ZOI). The zone of inhibition observed for the aqueous extract of leaves was 15.43 ± 0.30 mm for CA, 8.30 ± 0.15 mm for CT, and 13.90 ± 0.17 mm for CG. The ZOI for methanolic extract of leaves was 15.90 ± 0.56 mm for CA, 13.17 ± 0.09 mm for CT, and 14.57 ± 0.23 mm for CG. The ZOI for the aqueous extract of flowers was 14.30 ± 0.12 mm for CG, 12.33 ± 0.18 mm for CT, and 14.27 ± 0.50 mm for CA. The ZOI for methanolic extract of flowers was 15.27 ± 0.47 mm for CA, 10.90 ± 0.46 mm for CT, and 14.30 ± 0.47 mm for CG. Fluconazole (positive drug) showed maximum ZOI against CT (25.97 ± 0.49 mm), followed by CA (25.93 ± 0.54 mm), and CG (22.40 ± 0.31 mm). DMSO does not show ZOI against any of the strains (CT, CA, CG). The results of antifungal activity of extracts of S. obvallata against three fungal strains have been represented in Table 2.

GC-MS analyses of methanolic extracts of Saussurea obvallata

The results of GC-MS analyses of methanolic leaf and flower extracts of Saussurea obvallata showed the presence of 36 and 48 components, respectively based on separation of individual peaks through GC as per their retention time (Rt) and area per cent under individual peaks (Tables 3 & 4). The mass spectra of these compounds were matched with the spectra of known compounds listed in WILEY8.LIB and NIST08.LIB spectral databases/ libraries. Some of these components could not be identified by comparison using any of these libraries; such unidentified GC peaks numbered five (5; Rt: 9.113, 10.662, 10.887, 19.407, and 22.337 min) in the flower extract and one (1; Rt: 15.552 min) in the leaf extract. Most of the components presented in the extracts of leaves and flowers have been already reported in respect of different biological activities namely, Curumene (for anticancer), Methyl acetate (for hepatoprotective), Nerolidol acid (for antioxidant, anti-inflammatory and pesticide) and Piperine (malaria and respiratory disease), Stigmasterol (antioxidant, antimicrobial, anticancer) etc.
Table 3

Chemical composition of methanolic leaf extract of Saussurea obvallata

SN

Name of components

Mol. Formula

Area per cent (%)

1

Octyl cyanide

C9H17N

0.17

2

Benzeneacetaldehyde

C8H8O

0.10

3

Pyranone

C6H8O4

0.72

4

Pentadecane

C15H32

0.21

5

(RS)-nicotine

C10H14N2

0.07

6

Tetradecane

C14H30

0.27

7

Amantadine

C10H17N

0.18

8

Hexadecane

C16H34

0.16

9

α-Bisabol oxide B

C15H26O2

0.15

10

Blumenol C

C13H22O2

0.08

11

Tetradecanoic acid

C14H28O2

0.10

12

N-Acetylimidazole

C5H6N2O

0.15

13

Androsterone, trifluoroacetate

C21H29F3O3

0.08

14

Phytol

C20H40O

0.25

15

Diisobutyl phthalate

C16H22O4

0.06

16

Eltanolone

C21H34O2

11.43

17

Proximadiol

C15H28O2

0.24

18

Doconexent

C22H32O2

9.00

19

Palmitic acid

C16H32O2

11.84

20

Dehydrocostus lactone

C15H18O2

21.98

21

Strophanthidin

C23H32O6

0.25

22

n-Nonadecanol-1

C19H40O

0.11

23

Methyl linoleate

C19H34O2

0.26

24

Linoleic acid

C18H32O2

22.50

25

Stearic acid

C18H36O2

2.38

26

1-Docosanol

C22H46O

1.75

27

Methyl oleate

C19H36O2

0.09

28

Bupleuronol

C17H20O2

1.23

29

Andrographolide

C20H30O5

0.31

30

Nitrocyclododecane

C12H23NO2

0.16

31

1,2-Benzenedicarboxylic acid

C24H38O4

0.10

32

Stachydrine

C7H13NO2

0.37

33

Gama-Stearolactone

C18H34O2

0.91

34

Talaroconvolutin B

C32H43NO4

0.11

35

22-Tricosenoic acid

C23H44O2

0.68

36

Litsomentol

C30H52O2

2.25

Total identified compounds

90.70%

Unknown compounds

00.36%

Table 4

Chemical composition of methanolic extract of flowers of Saussurea obvallata

SN

Name of components

Mol. Formula

Area per cent

1

α –Terpineol

C10H18O

3.3

2

Methyl octanoate

C9H18O2

0.05

3

Cinnamaldehyde

C9H8O

0.44

4

Linalyl acetate

C12H20O2

4.94

5

Eugenol

C10H12O2

0.66

6

Methyl eugenol ether

C11H14O2

0.21

7

Curumene

C15H24

2.79

8

Nerolidol

C15H26O

0.28

9

n-Heptadecanol-1

C17H36O

0.08

10

Caryophyllene oxide

C15H24O

0.56

11

Geranyl linalool isomer b

C20H34O

0.16

12

n-Pentadecanol

C15H32O

0.53

13

Heptadecane

C17H36

0.59

14

Pentadecyclic acid

C15H30O2

0.42

15

Methyl palmitoleate

C17H32O2

0.34

16

Methyl palmitate

C17H34O2

12.18

17

Bicyclohexyl

C12H22

0.12

18

Palmitic acid

C16H32O2

4.65

19

Stearic acid

C18H36O2

0.43

20

Lignoceric acid

C24H48O2

0.50

21

Methyl stearate

C19H38O2

3.67

22

Camphor

C10H16O

0.96

23

Dibutyl sebacate

C18H34O4

1.04

24

Methyl linoleate

C19H34O2

0.31

25

Prodlure

C16H28O2

0.14

26

Menthyl acetate

C12H22O2

0.12

27

Rishitin

C14H22O2

0.07

28

1-Docosanol

C22H46O

0.21

29

Methyl linolenate

C19H32O2

0.14

30

Methyl oleate

C19H36O2

1.02

31

Henicosanoic acid

C21H42O2

2.24

32

Bornyl cinnamate

C19H24O2

0.19

33

Gama-Stearolactone

C18H34O2

0.12

34

Docosanoic acid

C22H44O2

0.18

35

Tridecanedial

C13H24O2

0.13

36

Gondoic acid

C20H38O2

2.92

37

Tricosanoic acid

C23H46O2

1.46

38

Deflazacortalcohol

C23H29NO5

0.10

39

Methyl nervonate

C25H48O2

0.15

40

Pentacosylic acid

C25H50O2

0.93

41

Squalene

C30H50

0.52

42

17-Pentatriacontene

C35H70

0.06

43

Eicosane

C20H42

0.11

44

Piperine

C17H19NO3

0.18

45

Nonacosane

C29H60

0.10

46

Stigmasterol

C29H48O

0.13

47

β- Sitosterol

C29H50O

0.17

48

Tetratetracontane

C44H90

0.10

Total identified compounds

50.70%

Unknown compounds

39.53%

The major components in the methanolic extract of leaves of S. obvallata were Linoleic acid (22.50%); followed by Dehydrocostus lactone (21.98%); Palmitic acid (11.84%); Eltanolone (11.43%); and Doconexent (9%). However, major components in methanolic flower extract of S. obvallata was not be identified by the known libraries (Rt: 19.40; 38.85%,); followed by Methyle palmitate (12.18%); Linalyl acetate (4.94%); Palmitic acid (4.65%); Methyl stearate (3067%). After GC-MS analyses of both the extracts of S. obvallata, few components in leaves and flowers extract have already been reported for various biological activities, such as anti-oxidant, anti-microbial, hemolytic, cancer preventive, chemo preventive, anti-tumor, immuno-stimulant, etc. (Table 5). Furthermore, other uses of these components are in perfume industry, and in the preparation of pesticides, anti-androgenic formulations, flavour additives, and for sunscreens. Six (6) components, namely Palmitic acid, Methyl linoeate, Stearic acid, 1-Docosanol, Methyl oleate, Gama-Stearolactone, are common in both the extracts. While some components being common in both the extracts have not been reported to exhibit any biological activity.
Table 5

Bioactive components present in the methanolic extracts of leaves and flowers of Saussurea obvallata

SN

Components

Biological activity

References

1

Benzeneacetaldehyde

Anti-microbial, anti-inflammatory.

[22]

2

Pyranone

Anti-microbial, anti-inflammatory, anti-oxidant, anti-proliferative.

[23]

3

Tetradecanoic acid

Anti-fungal, anti-oxidant, cancer-preventive, cosmetic.

[24]

4

Phytol

Anti-microbial, anti-inflammatory, anti-nociceptive.

[25]

5

Palmitic acid

Anti-androgenic, hemolytic, cancer preventive, sunscreen, perfumery.

[26]

6

Methyl linoleate

Hepatoprotective, anti-microbial.

[27]

7

Linoleic acid

Anti-inflammatory, anti-microbial.

[28, 29]

8

Gama-Stearolactone

Anti-tumor, anti-inflammatory, anti-convulsant activity.

[27, 3032]

9

α -Terpineol

Contractile activity; anti-bacterial; anti-microbial; Insecticidal.

[3236]

10

Cinnamaldehyde

Anti-cancer, anti-microbial, anti-oxidant

[37]

11

Curumene

Anti-tumor, anti-bacterial, anti-inflammatory, sedative, fungicide.

[3843]

12

Nerolidol

Anti-tumor, analgesic, anti-inflammatory, sedative, fungicide.

[44, 45]

13

n-Heptadecanol-1

Anti-microbial, anti-inflammatory.

[46, 47]

14

Caryophyllene oxide

Anti-microbial, anti-inflammatory.

[4852]

15

Pentacosylic acid

Anti-bacterial.

[27, 52, 53]

16

1-Docosanol

Anti-microbial, anti-herpetic agent, wounds, anti-herpes.

[5456]

17

Methyl acetate

Anti-oxidant, anti-microbial, anti-inflammatory and phytotoxic.

[57]

18

Piperine

Anti-oxidant, anti-microbial, anti-tumor, pesticide.

[58, 59]

19

β-Sitosterol

Anti-microbial, thyroid inhibitory, hypoglycemic effects.

[60, 61]

20

Stigmasterol

Anti-oxidant, thyroid inhibitory, hypoglycemic effects.

[60, 61]

21

Squalene

Anti-oxidant, anti-microbial, cancer preventive, pesticide.

[62]

To the best of our knowledge this would appear to be the first report regarding detailed phytochemical analysis, medicinal and anti-microbial properties, and discussion on the bioactive components of S. obvallata. During the present study, basic information has been generated about total phenolic & flavonoid contents, antioxidant activity (using DPPH & H2O2 assays), and antimicrobial activity. GC-MS analyses were carried out to elucidate the chemical composition of S. obvallata leaf and flower extracts (methanolic). The composition of plant extracts depend on their origin, extraction techniques used, the time and temperature of extraction, nature of solvent used, its concentration and polarity, quantity and the secondary metabolite composition of a given extract [63]. Variations in the extraction methods like the duration of the extraction period, the solvent used, pH, temperature, particle size, and the solvent-to-sample ratio [64] can lead to different results. Phytochemical investigations of S. obvallata extracts showed that, as expected, it contains different types of constituents like alkaloids, flavonoids, terpenoids, glycosides, saponins, etc. using different solvents, e.g., methanol, ethanol, chloroform, and distilled water as solvents [11]. Similar pattern and constituents were reported by Alaagib & Ayoub [65] for Saussurea lappa.

Phenolic and flavonoid compounds are produced by plants and are known to have strong anti-oxidant activity. They exert a positive effect on both plants and human health, and some derivatives of flavonoids possess antibacterial activity against a range of microbes. In the present study the maximum and minimum values for TPC were recorded for methanolic leaf extract and aqueous floral extract (358.67 ± 0.17 and 132.67 ± 0.17 mg GAE g− 1 dw, respectively) of S. obvallata, while maximum TFC (326.88 ± 2.11 mg QE g− 1 dw) was found in the aqueous floral extract, and the minimum TFC (65.73 ± 1.55 mg QE g− 1 dw) was found in the methanolic extract of flowers. On the other hand, Saussurea involucrata was reported to possess significant anti-oxidant activity (high phenol and flavonoid content) and is known to be used by a large population in China for medicinal purpose [66]. Phenolic and flavonoid contents in Saussurea obvallata are in general agreement with the previous findings on other species of Saussurea.

Free radicals can cause damage to essential proteins, DNA, lipids and also be attributed to various human diseases such as cancer, cardiovascular diseases, neurodegenerative disorders, etc. due to oxidative stress. The most important role of antioxidants is to suppress free radical mediated oxidation by inhibiting the production of free radicals through scavenging activity. Ascorbic acid was used as a standard antioxidant compound for DPPH assay, and H2O2 scavenging assay. Maximum per cent DPPH scavenging activity (82.88 ± 0.48%) was recorded in the methanolic extract of flowers, and the minimum (29.25 ± 0.86%) in the aqueous extract of flowers in S. obvallata. The maximum per cent H2O2 scavenging activity (41.05 ± 0.46%) was recorded in the methanolic leaf extract, and the minimum (39.75 ± 0.36%) in the aqueous leaf extract at 1 mg mL− 1 concentration of extracts. The same pattern in respect of antioxidant activity by DPPH assay was observed by Yao et al. [67] from two polysaccharides (SCIP1–2 and CSIP2–3) isolated from Saussurea involucrata, and they reported 60.3% and 88.7% DPPH scavenging activity at a dose of 8 mg mL− 1, respectively for the two polysaccharides. S. obvallata therefore contains more antioxidant potential compared to S. involucrata. In this test, it was observed that methanol as a solvent resulted in higher values for TPC, DPPH and H2O2 assays, while the aqueous extract resulted in higher values for TFC.

Nowadays, increased risk of infection and resistance against antibiotics has become a persistent and wide spread problem and antibiotics are often used indiscriminately against diseases causes by various microbes. Thus, it is an important task for investigators to be constantly in the lookout for an alternate chemical agent against theses human pathogens. In this study, four bacterial strains and three fungal strains were used for the analyses of antimicrobial activity of S. obvallata extracts. For aqueous leaf extract, the maximum ZOI (20.43 ± 0.30 mm) was found in respect of S. aureus while the minimum ZOI (11.57 ± 0.23 mm) was associated with E. coli. The maximum and minimum ZOIs in respect of methanolic leaf extract were 19.90 ± 0.56 mm for K. pneumoniae and 10.83 ± 0.43 mm for E. coli, respectively. The aqueous extract of flowers showed maximum ZOI (20.50 ± 0.26 mm) for P. aeruginosa and minimum ZOI (13.90 ± 0.21 mm) for E. coli. The methanolic extract of flowers caused maximum ZOI (16.33 ± 0.17 mm) for P. aeruginosa and minimum ZOI (08.87 ± 0.19 mm) for E. coli. In general, it was observed using this test that E. coli exhibited resistance against both the extracts (lower ZOI).

In the case of antifungal activity of S. obvallata extracts, maximum ZOI (15.43 ± 0.30 mm) was recorded in respect of C. albicans and the minimum ZOI (08.30 ± 0.15) was recorded for C. tropicalis. However, maximum ZOI (15.90 ± 0.56 mm) in respect of C. albicans and the minimum ZOI (13.17 ± 0.09 mm) in case of C. tropicalis were recorded in methanolic extract of leaves. Aqueous floral extract exhibited maximum ZOI (14.30 ± 0.12 mm) for C. glabrata and minimum ZOI (12.33 ± 0.18 mm) for C. tropicalis, while the maximum ZOI (15.27 ± 0.47 mm) and minimum ZOI (10.90 ± 0.46 mm) were found in respect of C. tropicalis. In this experiment, it has been shown that C. tropicalis exhibited resistance against S. obvallata extracts (lower ZOI). Khalid et al. [68] have reported antimicrobial activity of S. lappa root extract against three Gram positive and two Gram negative bacteria. Methanol, hot water and cold water were used for the extraction, and the range of zone of inhibition was observed between 10 and 18 mm for methanolic extract, 8–11 mm for hot water extract and 9–13 mm for cold water extract.

Antimicrobial activity of methanolic and chloroform extracts of S. lappa against E. coli, P. aeruginosa, K. pneumoniae, P. vulgaris, S. aureus, C. albicans, A. niger were reported by Thara & Zuhra [69]. Both the extracts of S. lappa exhibited significant activity against all bacterial strains, but some fungal strains exhibited lower zone of inhibition. Antimicrobial activity of Saussurea lappa (roots) was reported by Negi et al. [70] the samples were collected from four different regions of Uttarakhand and methanol was used for the extraction. Negi et al. [70] tested six bacterial strains (S. typhimurium, E. coli, C. freundii, P. vulgaris, E. faecalis and S. aureus). The ZOIs were 6–8 mm for E. coli, 5–10 mm for C. freundii, 5–8 mm for E. faecalis and 6–10 mm for S. aureus. The extract of S. lappa exhibited excellent activity against these four bacterial strains compared to a known standard used. Two bacterial strains, Proteus vulgaris and S. typhimurium showed resistance against the root extract up to 100 mg mL− 1 concentrations. Alaagib et al. [65] also reported antibacterial activity of S. lappa (black root) extract against five bacterial strains (B. subtilis, S. aureus, E. coli, P. aeruginosa and K. pneumoniae). Petroleum ether, chloroform, methanol, and water were used for extraction, and all five strains exhibited variable activity, Gram positive bacteria were more sensitive to the extract in comparison to Gram negative bacteria. The ZOIs caused by the root extract of S. lappa ranged from 11 to 21 mm (petroleum ether), 11–22 mm (chloroform), 11–17 mm (methanol), and 11–15 mm (water) extract. In respect of E. coli the zone of inhibition formed was quite low as also in the present study.

GC-MS analyses helped in the elucidation of chemical composition of both the extracts (leaves and flowers) of S. obvallata. Through these analyses thirty six (36) components were found to be present in the leaf extract, and forty eight (48) components in the flower extract. One (1) component in the leaf extract and five (5) components in the flower extract could not identified by known libraries. Total 21 components were enumerated as bioactive components in the two extracts based on previous studies, while 6 components were common in respect of both the extracts [71]. Many of the reported bioactive components were had been isolated from various sources, and already known for anti-oxidant and anti-microbial activity, etc. [72]. Thus, it can be concluded that the anti-oxidant and anti-microbial activities along with other medicinal properties associated with this plant may also be caused by theses bioactive components.

Conclusions

This study provides a fairly comprehensive report on TPC, TFC, anti-oxidant and anti-microbial activities, and GC-MS analyses of leaf and flower extracts of Saussurea obvallata. The results of present investigation are in general agreement with earlier studies on anti-oxidant, anti-microbial, and GC-MS analyses of other species of Saussurea genus. These results may provide a strong basis for traditionally known medicinal properties of S. obvallata, many of which are also mentioned in India holy books. Screening and identification of components and systematic evaluation of their medicinal properties may provide clues for a potential drug, and its other bioactivities may provide ground for these to be recommended for further investigations.

Abbreviations

DMSO: 

Dimethyl sulfoxide

DPPH: 

2,2-diphenyl-1-picrylhydrazyl

GC-MS: 

Gas Chromatograpy – Mass Spectrometry

H2O2

Hydrogen peroxide

TFC: 

Total flavonoids content

TPC: 

Total phenolic content

Declarations

Acknowledgements

The authors wish to thank Prof. L.M.S. Palni, Prof. Ashish Thapliyal and Prof. Susheel Verma for providing facilities and encouragement. Help and support received from the Graphic Era (Deemed to be University), Dehradun, India and Jawahar Lal Nehru University, Delhi, India are gratefully acknowledged. We also thank Dr. Priyanka Sati for providing help during Statistical analysis.

Funding

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this article.

Authors’ contributions

PS collected the samples, performed all the experiments, and drafting the manuscript with SP. Both authors read the manuscript and approved the final version.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Department of Biotechnology, Graphic Era (Deemed to be University), 566/6, Bell Road, Clement Town, Dehradun, Uttarakhand, 248 002, India

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