Preparation and Characterization of Extract

The collection of the plant was carried out from the Himalayan region of Dharamshala (Naddi), Himachal Pradesh, India and identification was carried out at the University school of Agricultural Sciences (RBU, Chandigarh) under voucher with Ref. No. RBU/USAS/HOD/22/190. Young and healthy leaves of A. tortuosum were selected and firstly cleaned by washing with tap water and afterward with demineralized water to expel the residue and dust particles over their surface. After draining free water, the material was dried under the shade at the room temperature for 30 days and ground to obtain the powdered form. 100 g of the powdered sample was processed for 12 hrs. By the Soxhlet extraction process utilizing 300 mL of methanol as a solvent at its boiling point ^[16]. The solvent was removed from the extract by using a rotary vacuum evaporator. The obtained final product was characterized by gas chromatography-mass spectroscopy (GC-MS) analysis (Model THERMO Scientific Trace 1300GC) using the capillary column (Model, Restek USA RTx-5Sil MS) with dimensions of 30 m × 0.25 mm × 0.25 mm. Helium was delivered at a flow rate of 1 mL/min. as a carrier at a split ratio of 1:5. The mass spectra were acquired utilising the electron ionisation (EI) mode with an energy of 70 electron volts (eV) at a spectral range (m/z) of 40-700. The experimental setup involved setting the interface temperature to 280 °C and the ion source temperature to 200 °C. The identification of different constituents was conducted through the comparison of their retention time and mass spectral data with the Wiley8 and NIST11 library spectral database. The quantification of the compounds was performed by analysing the area under the peak, and the resulting data was expressed as a percentage. The analysis of each sample was conducted in triplicate.

Total Phenolic Content

The Folin-Ciocalteu spectrophotometric method was used for the quantitative determination of the total phenolic content of the extract as described by Rebaya et al.^[17]. 1 mL of extract (1 mg/mL) or standard gallic acid solution (30-150 mg/L) was diluted up to 10 mL by adding deionized water followed by addition of Folin-Ciocalteu reagent solution (1 mL). After the addition of 2 mL of sodium carbonate solution, 20% (w/v), the mixture was kept in dark for 60 min. and the absorbance was measured at 750 nm (UV-Visible spectrophotometer, Agilent Cary 60). The calibration curve provided the estimate of the total phenolic content (mg of gallic acid equivalent per g of dry extract weight).

Total Flavonoid Content

Aluminum chloride colorimetric technique was used to assess the TFC present in the extract as outlined by Stoyanova et al.^[18]. 1 mL of extract (1 mg/mL) or standard Rutin trihydrate solution (60-300 mg/L) was diluted up to 5 mL by adding deionized water. After adding 0.3 mL of NaNO₂ solution, 5% (w/v), the mixture was left untouched for 5 min. In this mixture, 0.3 mL of AlCl₃ solution, 10% (w/v) and 2 mL of 1 M NaOH were added followed by dilution to 10 mL with deionized water. The mixture was again kept in dark for 30 minutes at 25 °C and the absorbance at 510 nm was recorded (UV-Visible spectrophotometer, Agilent Cary 60). The total flavonoid content (mg of rutin trihydride equivalent per g of dry extract weight) was estimated from the calibration curve.

Antioxidant potential (DPPH Assay)

The antioxidant potential of the extract was quantitatively determined as free radical scavenging assay by using the DPPH method as given by Garcia et al.^[19]. 3 mL of fresh DPPH solution (0.1 mM) was poured into 0.2 mL of extract (1 mg/mL) and kept in dark for 30 minutes. The absorbance was recorded by Agilent Cary 60 UV-Visible spectrophotometer at 517 nm. The scavenging impact of the extract against DPPH free radical was determined using Ascorbic acid as a standard on the basis of the equation given below.

Where A_test and A_control are the absorbances of test sample/ standard solution and control respectively.

Anti-inflammatory Potential (β-glucuronidase inhibition assay)

As per the method given by Otang-Mteng et al.^[1], 0.1 mL of the extract, with varying dosage from 25-100 mg/mL, was preincubated for 5 min. in 0.1 M acetate buffer (pH 7.4) at 37 °C ^[1]. This mixture was further kept in dark for 30 min. after addition of 0.1 mL β-glucuronidase solution and 2 mL of NaOH (0.5 N) was added to terminate the reaction. A reference, Salicylic acid, was utilized with a concentration of 1 mM and the absorbance of the solution was recorded at 410 nm (UV-Visible spectrophotometer, Agilent Cary 60).

GC-MS analysis

Figure 1 illustrates the existence of various secondary metabolites or phytochemicals in the GC-MS profile of the extract. A total of fifty-three phytochemicals were detected in the chromatograph of the methanolic leaf extract of A. tortuosum as shown in Table 1. Nonane was eluted first with a retention time of 4.064 min. in the leaf extract. The major peak area was obtained for 6, 10, 14-trimethyl-2-pentadecanone as 54.55% and phytol as 18.86% in the leaf extract. The other identified major phytochemicals included 2-Pentadecanone, 6,10,14-trimethyl-, Phytol, Benzene, 1,3-dimethyl-, 2-Pentanone, 4-hydroxy-4-methyl-, Cyclohexane, (2-methylpropyl)-, Cyclohexane, 1-methyl-2-propyl-, Cyclohexane, butyl-, Naphthalene, decahydro-, trans-, Undecane and hexadecamethyl cyclooctasiloxane. The results confirmed the presence of important bioactive compounds like terpenoids, polyphenols, hydrocarbons and organosiloxanes in the leaf extract of A. tortuosum. Some of these phytochemicals with significant biopotential have been listed in Table 2.

Figure 1 GC-MS analysis of leaf extract of A. tortuosum. 

Total Phenolic Content (TPC)

The total phenolic content is the quantitative approach to determine the amount of polyphenols present in the plant. Phenols are one of the important plant constituents as the radical scavenging ability depends on the hydroxyl group ^[12]. The phenolic groups are secondary metabolites and have redox properties that allow them to function as a radical scavenger in various natural activities such as antioxidant, antibacterial and antifungal ^[20]. The total phenolic content also depends upon the nature of the extraction method and solvent used. The quantity of phenolic content available in methanolic leaf extract of A. tortuosum was calculated from a calibration curve. The TPC for leaf extract of A. tortuosum was found as 171.05 gallic acid equivalents/g. The high phenolic content of the plant extract has been correlated with the high antioxidant activity of the medicinal plants in various reports ^{[21, 22]}.

Total Flavonoid Content (TFC)

Flavonoids such as flavanols and flavones are secondary plant metabolites that are more soluble in polar solvents as compared to the nonpolar solvent, due to this methanol extract has higher flavonoid content as compared to the other extract ^[17]. These metabolites are responsible for the color as well as antioxidant, anti-inflammatory and antimicrobial activity of plants. The antioxidant activity of the extract is also found to be dependent upon the quantity and position of hydroxyl groups in the flavonoids. The amount of flavonoid present in the leaf extract of A. tortuosum was also measured by the regression calibration curve. TFC for the leaf extract was found as 183.5 rutin trihydride equivalents/g. A similar result was reported previously in the case of tuber extract of A. tortuosum^[5].

Antioxidant Assay

The polyphenolic compounds present in medicinal plants are referred to as antioxidant agents and used as radical scavengers or metal chelators ^[16]. The antioxidant effect of the plant depends on phytochemicals present as both major and minor constituents that play an effective role in DPPH radical activity. Literature reports different methods for evaluating the antioxidant activity dependent upon free radical scavenging activity having different mechanisms ^[23-25].

The general mechanism given for DPPH activity has been illustrated in Figure 2 ^[25]. In this examination, the leaf extract of A. tortuosum was found to exhibit significant DPPH scavenging activity with IC₅₀ value as 936 μg/mL. The results have been found consistent with similar studies carried out on the tuber extract of A. tortuosum with scavenging activity against stable DPPH radical having IC₅₀ value of 852 μg/mL ^[5].

Figure 2 Illustrative mechanism for DPPH scavenging activity. 

Table 1 GC-MS analysis of the leaf extract of A. tortuosum 

Table 2 Structure and activities of major phytocompounds reported in GC-MS analysis 

β-Glucuronidase inhibition potential

β-glucuronidase, an enzyme found in neutrophil lysosomal membranes, has been identified as one of the mediators of the inflammatory response ^[5]. Hence, the anti-inflammatory potential of leaf extract was determined in terms of β-glucuronidase inhibition. A substantial inhibitory action against the β-glucuronidase enzyme in a concentration-dependent manner was observed, with the highest inhibition observed at a concentration of 100 mg/mL in comparison to the control (salicyclic acid). 82.36% inhibition was observed for the extract (IC₅₀ value as 37.45 mg/mL) as compared to the 72.81% inhibition by the control. Thus, A. tortuosum leaf extract exhibited a greater ability to inhibit β-glucuronidase activity than an established anti-inflammatory agent, salicylic acid ^[26].