INTRODUCTION:

P. kurroa is a high-altitude herb, found in India, Nepal, Pakistan, and China of the Himalayan region, at a height of 3000 to 5000m in India¹. TheHigh Altitude Plant Physiology Research Centre (HAPPRC) has designed the farmtechniques to grow P. kurroa in Uttarakhand². It is widely employed in both the ISM (Indian system of medicine) and modern pharmaceutical industries³. P. kurroa juice is bitter, used against various diseases, and a bitter tonic almost as efficacious as Gentian, digestive, stomachic, and febrifuge.

It also exerts a stimulating influence on the mouth’s gustatory relationship with the sensation of taste nerves and finally results in enhanced stomach juice secretion⁴. The rhizomes extract of P. kurroa is used as an antioxidant, anti-inflammatory, hepatoprotective effect, anticholestatic, antioxidant, and immunomodulatory activity. P. kurroa is widely used to cure various ailments namely leukoderma, anti-inflammatory, jaundice, fever, and urinary diseases, antineoplastic activity, chronic fevers, treatment of dyspepsia, diarrhea, and scorpion sting⁵. Other plants like artemisia were also found effective against various diseases⁶. In 2013 this plant has been declared endangered status in a prioritization workshop on sustainability for plant species in the Jammu and Kashmir, Himachal Pradesh, and Uttarakhand states of Northwest Himalayan (India)⁷. This plant possesses various biologically important natural products. This plant needs to be preserved because of its importance of the plant. In this article, we have reported regarding the identification and quantification of six natural compounds from Picrorhiza kurroa leaf extract and their biological assessment regarding antibacterial and antioxidant interest⁸.

MATERIALS AND METHODS:

The plant sample was air-dried in the darkroom to avoid overlosses of thermolabile and volatile constituents.The plant material was reduced to a coarse fine powder with the help of a mechanical grinder⁹. Solvent selection for the extract preparation was done in increasing polarity viz. n-hexane, chloroform, ethylacetate, acetone, n-butanol, methanol, hydroalcoholic, and water¹⁰. Extraction was done using NCI protocol^11,12. Eight different extracts were collected such as n-hexane, chloroform, ethyl acetate, acetone, n-butanol, methanol, hydroalcoholic and aqueous extract. The extracts were used for the antibacterial and antioxidant studies^13,14.

Liquid chromatography-mass spectroscopy:

The instrument used for LC-MS was Micro TOFQ ESI. For the liquid chromatography separation, Phenomenex C18 (150 × 4mm i.d., 5µ) with a single quadrupole MS analyzer was used. mobile phase, 0.5 percent formic acid–acetonitrile (75:25 percent) was used 0.5ml/min was the flow rate¹⁵. The solvent had isocratic elution controlled. The temperature of the column was maintained at 30°C. In the positive ion mode, the MS spectrum was acquired and 50m/z-1000m/z was scanned by using a spectrum database of organic compounds, and mass fragmentations were identified¹⁶.

HPTLC quantification of major constituents:

The major components of Picrorhiza kurroa leave DCM/ methanol (1:1) extract were quantified using an HPTLC instrument with automatic sample applicator CAMAG Linomat V, TLC scanner III, Camag twin trough chamber 10×10cm, and WinCATS software in the present study. The extract and standard solutions suspended in methanol were applied in the form of a band having a band with a width of 8 mm; a distance between 14mm bands and a constant application rate of 150nL s-1 using a micro-syringe (Hamilton-Bonaduz Schweiz, Linomat syringe, size 500µl) to 60 F254 TLC (10 × 10cm with a thickness 200µm) silica gelpre-coated plates. The calibration curve was drawn. metabolites were investigated in TLC with regressionfrom the calibration plot and expressed as % w/w.^17,18

RESULT:

In LC-MS mass analysis of the highly polar DCM:Methanol/1:1 extract of Picrorhiza kurroa. The chemical constituents present in the DCM:Methanol/ 1:1extract were recognized using LC-MS spectroscopy. Numerous peaks and analyzation of each peak were done further. When compared with masses of several natural compounds in the databases (Dictionary of Natural Product 28.2 and others mentioned in methodology), we found it, almost similar with Quercetin, caffeic acid, α-sitosterol, catechin, lupeol, etc. The discrepancy of this method was same in masses (Masses of α-carotene, and lycopene, Vanillic acid and Gallic acid, etc). Further authentication was carried out using HPLC.

Qualitative HPLC analysis of alcohol:DCM/ 1:1 extract of Picrorhiza kurroa was and our previous experiment by LCMS. The phytochemical screening was based on the observed chromatogram. Eighteen peaks were identified, which indicates the presence of major compounds in the extract. We supposed that the maximum area should correspond to catechin. The early assumption of similar masses bearing compounds identified from LCMS might be reduced or the instrument was unable to detect them due to less concentrations of those molecules in the sample solution. Different peaks, peak area, retention time, and percentagearea were identified. We were assured now of the presence of eighteen major compounds in alcohol:DCM/ 1:1 leaf extract of Picrorhiza kurroa.

HPTLC analysis of alcohol:DCM/ 1:1 leaf extract R_f value of the markers found to be match with six compounds in the herbal extract. We were unable to identify/quantify the other 12 compounds due to the non-availability of some markers or might be due to less concentration of those which we have installed the other markers. The identified compounds were quantified usingprotocol¹⁹. In the the Quantification of Aucubinthe mobile phase consisting of ethyl acetate: Formic acid: Methanol: Water (7.7:1.5:0.8 v/v) showed sharp peaks with R_f value of 0.20 for aucubin Figure 1(Fig A). Aucubinfound in the extract was 0.63% w/w of extract.

Fig. 1. (A) TLC (B) HPTLC of the extract with aucubin (C) Std Calibration graph.

Fig. 2.(A) TLC (B) HPTLC of the extract with β-Sitosterol (C) Std Calibration graph.

Fig. 3(A) TLC (B) HPTLC of the extract with chlorogenic acid (C) Std Calibration graph.

Fig. 4(A) TLC (B) HPTLC of the extract with Ellagic acid (C) Std Calibration graph.

Fig. 5 (A) TLC (B) HPTLC of the extract with diosgenin (C) Standard Calibration graph.

Fig. 6(A) TLC (B) HPTLC of the extract with kutkin (C) Standard Calibration graph.

For the Quantification of β-Sitosterol The mobile phase n-hexane: ethyl acetate (8:2, v/v) showed sharp peaks with Rf value of 0.66 for β-Sitosterol Figure 2(Fig A). β-Sitosterol found in the extract was 0.70% w/w of extract. In the Quantification of Chlorogenic acid: The Mobile phase consisting n-hexane: ethyl acetate (10:1.1:1.1:0.5, v/v) showed sharp peaks with R_f value of 0.59 for chlorogenic acid figure 3(Fig. A). Chlorogenic acid found in the extract was 2.70% w/w of extract. Quantification of Ellagic acid Mobile phase consisting Toluene: Ethyl acetate: Formic acid (5:4:1, v/v) showed sharp peaks with R_f value of 0.20 for ellagic acidfigure 4(Fig. A). Ellagic acid found in the extract was 0.50% w/w of extract. Quantification of Diosgenin: The Mobile phase n-hexane: Ethyl acetate (8:2,v/v) showed sharp peaks with R_f value of 0.36 for diosgenin Figure 5 (Fig. A). Diosgenin found in the extract was 2.70% w/w of extract. Quantification of Kutkin The Mobile phase Ethyl acetate: Formic acid: Methanol (6:0.6:0.4, v/v) showed sharp peaks with R_f value of 0. 44 and 0.66 for kutkin figure 6 (Fig A). Kutkin found in the extract was 2.70% w/w of extract.

Antibacterial Activity:

The prepared eight leaf extracts were evaluated against five bacterial strains at a concentration of 100μl/well^20-22. Chloramphenicol (HiMedia) as a positive control. DMSO was used as a negative control in each agar plate to make sure that the solvent used for dissolving the extract does not show antibacterial activity^23,24. The zone of inhibition was measured in millimeters. The graphical representation of the antibacterial activity is shown in figure 7.

Fig. 7Antibacterial activity of the plant extract

In the Petri plate, 1, 2, 3, 4, 5, 6, 7, 8 is aqueous, n-hexane, methanol, chloroform, ethyl acetate, acetone, n-butanol, hydroalcoholic extracts respectively which are brown. Here maximum potency for antibacterial activity was shown by acetone extract (11.66±1.52) and minimum by aqueous extract (8.00±1.00) against Staphylococcus aureus test bacteria. In the case of Staphylococcus mutans maximum potency for antibacterial activity is shown by acetone extract (12.01±1.00) and minimum by chloroform extract (9.33±0.57) against Staphylococcus mutans test bacteria. Against Escherichia coli the maximum antibacterial activity was shown by acetone extract (11.66±0.42) and minimum by chloroform extract (9.00±1.00) against Escherichia coli test bacteria. Here maximum potency for antibacterial activity was shown by acetone extract (11.00±1.73) and minimum by petroleum ether extract (0.00±0.00) against Pseudomonas areuginosa test bacteria. Where maximum potency for antibacterial activity was shown by acetone extract (11.33±1.52) and minimum by n-butanol extract (9.00±1.00) against Klebsiella pneumonia test bacteria.

Antioxidant activity:

Eight extracts and positive control (Ascorbic acid) were used for antioxidant activity using the DPPH method by known protocol.^25,26No extract was shown more potency than ascorbic acid but within all extracts, methanolic extract reported maximum potency (41.3±3.1µg/ml) for antioxidant activity and the minimum value was observed for hexane extract (389.5±1.3µg/ml). The increasing order of the extracts with respect to antioxidant activity are n-hexane < Aqueous < Chloroform < Hydroalcoholic < Acetone<n-butanol <Ethyl acetate< Methanol and is shown in table 1

Table 1: Scavenging activity IC₅₀ Value of P. kurroa of different extracts

Extracts	Activity	Acetone	59.3±3.6
n-hexane	389.5±1.3	n-butanol	48.2±4.3
Chloroform	84.2±3.4	Methanol	41.3±3.1
Ethyl acetate	46.2±1.4	Aqueous	159.5±1.6
Hydroalcoholic	103.2±2.8	Ascorbic acid	17.9±2.1

N.B. Antioxidant activity expressed as IC₅₀Value in µg/ml±S.D of three replicates.

DISCUSSION:

Six natural compounds including quercetin, caffeic acid, β-sitosterol, catechin, lupeol, and p-coumaric acid were identified from HPLC, mass spectroscopic analysis, and quantified from HPTLC analysis. MS investigation presumed the occurrence of twenty-one different natural compounds. The primary empathy of the compounds was thru from natural product databases. Secondary identification using HPLC repossessed the incidence of nineteen different natural compounds²⁷. HPTLC analysis established and quantified the presence of six major compounds. The amount of Catechin was found to be maximum, which was 2.86% w/w of DCM/Methanol leaf extract followed by Caffeic acid (2.06% w/w of extract). Among the eight different extracts hydroalcoholic extract showed good potency in terms of zone of inhibition), which was 14.33±0.25mm against S. aureus bacterial strain, acetone extract showed good potency (16.01±0.81mm) against S. mutans bacterial strain, ethyl acetate extracts showed good potency (16.33±0.47mm) against E. coli, ethyl acetate extracts showed good potency (17.35±0.58mm) against K. pneumoniae and n-hexane extracts showed good potency (16.33±0.38) against P. aeruginosa. The zone of inhibition for chloramphenicol ranged from 22.36 ±0.40 to24.66±0.46 against five strains of bacteria, which concludes that the leaf extracts of Picrorhiza kurroa possessed good antibacterial activity. Antioxidant activity was done by the DPPH method. IC₅₀values were calculated from the known protocol. Methanol extract showed good antioxidant activity (51.3 ±3.1µg/ml) and n-hexane showed the least (189.5±1.30 µg/ml). The IC₅₀ value of the control (Ascorbic acid) was17.9±2.1µg/ml, which concludes that the leaf extract is average concerning antioxidant activities.

CONCLUSION:

Picrorhiza kurooa plant was selected for the study. Six compounds were identified and quantified from plant extracts. Primary identification was done by HPLC & HPTLC. Antibacterial and antioxidant activity was examined for eight extracts of the plant. From HPLC total of 18 compounds and HPTLC total of six compounds were identified and quantified. For antibacterial activity among its eight extracts acetone extract showed good potency in terms of zone of inhibition in millimeters (11.66±1.52) against S. aureus, n-butanol showed (12.01±1.00) against S. mutans, acetone extracts showed (11.66±1.52) against E. coli, acetone extracts showed (11.33±1.52) against K. pneumoniae and acetone extracts showed (11.00±1.00) against P. aeruginosa. Chloramphenicol was used as a control for the antibacterial study for comparisons. The antioxidant activity assays of all the extracts were found to be average against ascorbic acid.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We are very thankful to Dr. Kulwant Rai Sharma (Professor and Head) for the authentication of the plant from the Department of Forest Product, DR YS Parmar University of Horticulture and Forestry, Nauni, Solan, H.P, India. We are very thankful for Himachal Pradesh State Biodiversity Board Project (HPSBB/F (16)-01/2014-3530) for providing financial assistance to do research work related to Picrorhiza kurroa plant.

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Received on 19.09.2021 Modified on 14.11.2021

Research J. Pharm. and Tech 2022; 15(12):5774-5778.

DOI: 10.52711/0974-360X.2022.00974