Major phenolic compounds, antioxidant, antimicrobial, and cytotoxic activities of Selinum carvifolia (L.) collected from different altitudes in India

Ravi Prakash Srivastava; Sachin Kumar; Lav Singh; Mayank Madhukar; Nitesh Singh; Gauri Saxena; Shivaraman Pandey; Arpit Singh; Hari Prasad Devkota; Praveen C Verma; Shatrughan Shiva; Sumira Malik; Sarvesh Rustagi

doi:10.3389/fnut.2023.1180225

. 2023 Jul 13;10:1180225. doi: 10.3389/fnut.2023.1180225

Major phenolic compounds, antioxidant, antimicrobial, and cytotoxic activities of Selinum carvifolia (L.) collected from different altitudes in India

Ravi Prakash Srivastava ^1,^†, Sachin Kumar ², Lav Singh ³, Mayank Madhukar ⁴, Nitesh Singh ^5,^†, Gauri Saxena ^1,^*,^†, Shivaraman Pandey ¹, Arpit Singh ¹, Hari Prasad Devkota ⁶, Praveen C Verma ⁷, Shatrughan Shiva ⁷, Sumira Malik ⁸, Sarvesh Rustagi ⁹

PMCID: PMC10382142 PMID: 37521418

Abstract

Antibiotic resistance poses a serious threat to public health, raising the number of diseases in the community. Recent research has shown that plant-derived phenolic compounds have strong antimicrobial, antifungal, and cytotoxic properties against a variety of microorganisms and work as great antioxidants in such treatments. The goal of the current work is to evaluate the anticancerous, antibacterial, antifungal, antioxidant, and cytotoxicity activities in the extracts of the different plant parts (leaves, stems, and roots) of S. carvifolia (L.) L. This is a medicinally important plant and has been used for different kinds of diseases and ailments such as hysteria and seizures. The phenolic compounds from the different plant parts were analyzed using HPLC and the following were found to be present: chlorogenic acid, gallic acid, rutin, syringic acid, vanillic acid, cinnamic acid, caffeic acid, and protocatechuic acid. Gallic acid was found to have the highest concentration (13.93 mg/g), while chlorogenic acid (0.25 mg/g) had the lowest. The maximum TPC value, which ranged from 33.79 to 57.95 mg GAE/g dry extract weight, was found in the stem. Root extract with 9.4 mg RE/g had the greatest TFC level. In the leaf and stem extracts, the RSC ranged from 0.747 mg/mL to 0.734 mg/1 mL GE/g dry extract weight, respectively. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to measure in vitro antioxidant activity. In a concentration-dependent way, promising antioxidant activity was reported. Moreover, 3,5-dinitrosalicylic acid (DNSA) and the Folin–Ciocalteu phenol reagent technique were used to determine reducing sugar content and total phenolic content, respectively. Antibacterial activity against eight strains (MIC: 250–1,000 μg/mL) was analyzed, and the stem extract exhibited maximum activity. Antifungal activity was also assessed, and potent activity was reported especially in the extract obtained from the stem. Cytotoxicity was evaluated using an MTT assay in the A549 cell line, where different doses (0.0625, 0.125, 0.25, 0.5, and 1 mg/mL) of leaf, root, and stem extracts were used. Treatment with these extracts reduced the cell viability, indicating that S. carvifolia may possess anticancer potential, which can be of great therapeutic value.

Keywords: antimicrobial, antioxidant, HPLC, MTT assay, pharmacology, Selinum carvifolia

Introduction

A serious concern to the public’s health is the emergence of pathogenic bacteria and fungi that become resistant to synthetic antibacterial treatments over a period of time. This may result in an increased load of several antibiotic resistant bacteria and illnesses caused by them (1).

Most of the conventional antibiotics currently available for purchase have significant adverse effects on patients and have been responsible for the emergence of multiple drug resistance in pathogenic bacteria (2). Previous research has shown that bactericidal antibiotics such as quinolones and aminoglycosides have negative side effects, while lactams cause mitochondrial dysfunction and excessive production of reactive oxygen species (ROS) in mammalian cells, resulting in oxidative damage to DNA, proteins, and membrane lipids (3). Free radicals have also been linked to a number of diseases, including ischemic heart disease, diabetes, atherosclerosis, cancer, inflammation, and aging (4).

The defense against free radicals and harmful microbes is greatly aided by secondary plant metabolites such as phenols, flavonoids, and terpenoids (5). In order to tackle these resistant infections while avoiding or limiting the side effects associated with the consumption of synthetic antibiotics, there has been increasing interest in the identification of novel natural antimicrobial agents (6). As a result, combining antioxidant therapy with antibiotic medication appears to be a strategy to reduce or avoid these side effects. Plant-derived medicines cause few side effects, and they have a long history of use in folk medicine for the treatment of infectious diseases and oxidative stress conditions (7, 8).

The use of antioxidant and antibacterial drugs together helps to boost their antioxidant and antibacterial capabilities. It has also been observed that phenols, flavonoids, and terpenoids can increase the sensitivity of various bacteria to particular antibiotics (9–11).

The antibacterial and antioxidant properties of natural compounds from higher plants may prove to be a new source with a novel mechanism of action (12). As a result, there are three different levels of interaction at play: interaction with the outside cellular components, engagement with the cytoplasmic membrane, and interaction with cytoplasmic components. To exercise their antibacterial effects, natural compounds might interact with bacterial cells on one level or all three levels.

They may find innovative active principles to circumvent resistance mechanisms in multidrug-resistant microbes as a result of their thorough and systematic screening (13).

Many medicinally important plants can be found in the high altitude, alpine, and sub-alpine regions of Uttarakhand, Himachal Pradesh, and the surrounding areas of the Western Himalayas in India. The richness of medicinal plants in the Himalayas reflects its distinct geological, topographical, ecological, climatic, and physiographic position (14, 15). These plants have ethnobotanical significance and are often utilized in traditional medicine (16, 17). Medicinal plants are known to contain specific bioactive compounds that can inhibit microbial growth in the environment; therefore, they play a significant role in the creation of effective therapeutic treatments (18–20). Pharmacological industries have created a large number of antibiotics (21–23). Researchers have been investigating the antibacterial activity of medicinal plants as a result of the acceptance of traditional medicine as an alternative form of healthcare and the development of microbial resistance to current antibiotics (24–26).

Recent research has revealed that the antioxidant activity of plant products is mostly due to the presence of phenolic components including flavonoids, phenolic acids, tannins, and other phenolic compounds (27–29). Studies report that excessive free radicals are responsible for various pathological conditions such as asthma, arthritis, inflammation, neuro-degeneration, Parkinson's disease, and diabetes. Natural antioxidants have become the focus of a multitude of studies aimed at identifying sources of potentially safe, effective, and low-cost antioxidants (30, 31). Herbal medications that include free radical scavengers have been shown to have therapeutic value (32). To avoid the oxidation of the vulnerable substrate, plants create an astonishing array of antioxidant molecules such as carotenoids, flavonoids, ascorbic acid, and others. Antioxidants are commonly used in the food industry to prevent lipid peroxidation (33, 34). Plants include different iso-quinoline alkaloids that have antibacterial, anticancer, anti-inflammatory, adrenolytic, sympatholytic, and anti-acetylcholine esterase effects, according to chemical and pharmacological research (35, 36). The most efficient method for quantifying phenolic and flavonoid chemicals is HPLC analysis (37).

Selinum carvifolia plants have shown useful therapeutic effects, which are mostly dependent on the presence of phenols, flavonoids, and terpenoids. This is a perennial herb native to the high altitudes of Uttarakhand, Himachal Pradesh, and the neighboring regions of Indian Western Himalayas (38). This important species grows in humus-rich mountainous regions of the Himalayas between 2,200–4,000 m in states of Himachal Pradesh, Uttarakhand, and Sikkim in India and in neighboring countries such as Nepal and China (39). In India, the genus Selinum, also known as Bhutkeshi, is used as an insecticide, a nervine sedative with anti-spasmodic stimulating effects, and in the cure of constipation, menstruation, and digestion, among other things (40). A paste made from the leaves of S. carvifolia has been ethno-medically used for wound healing for centuries. The smoke produced from burning the roots of S. carvifolia is reported to repel insects, and the root decoction contains antimicrobial properties that can be used to cure coughs, colds, fevers, and body problems (41–43). However, due to their indiscriminate usage, the plants now need to be grown in vitro for their cultivation and preservation (44).

Previous studies on Selinum mainly focused on the volatile constituents of the different plant parts (45–49), but there are hardly any detailed reports on the non-volatile constituents in the leaves, stems, and roots of Selinum carvifolium. Recently, two novel compounds, bhutkesoside A (1) and bhutkesoside B, and 10 known compounds from the roots of the same plant have been reported from the leaves of Ligusticopsis wallichianum (50). The present study, therefore, aimed at quantifying and identifying the phytochemical constituents of the leaves, stems, and roots of S. carvifolia collected from three different altitudes (2,150, 2,593, and 3,178 m) in the Chopta region of Uttarakhand, India, and probing their antioxidant activity, reducing sugar capacity (RSC), and antibacterial and cytotoxicity properties in order to establish the therapeutic potential of this plant.

Materials and methods

Collection of plant material

The samples were collected in September 2018 from three different altitudes: 2,150 m (latitude 22°21′12.4″N75°12′01.4″E), 2,593 m (latitude 25°23′14.9”N75°11′01.3″E), and 3,178 m (latitude 28°24′25.9″N 75°10′57.6″E). Five to six whole plants were collected from each altitude in the Chopta region of Uttarakhand, India. A GPS positioning system was used for geographic coordinates, and the elevation information of each measurement point was recorded. The plants were identified by Dr. L. B. Chaudhary, Principal Scientist, Herbarium (Angiosperm Taxonomy), National Botanical Research Institute, Lucknow, India, and the samples were deposited in the NBRI repository as voucher specimen LWG 105543.

HPLC

Analysis sample preparation

Ten grams of dry weight of raw material (roots, leaves, and stems) were powdered and poured into a conical flask. One hundred milliliters of 70% MeOH was added to it, and the extract thus formed was left for 48 h. Fractionation was performed in different solvents in the order of hexane (Hx), petroleum ether (PTE), and chloroform (Chl), as per the extraction methods of Abubakar and Haque (51). The residue was returned to the conical flask after the extracts from various plant sections were filtered through Whatman filter paper No.1 into a 250 mL volumetric flask. The extraction technique was performed two more times, with 70% methanol used to make up the difference in volume. Before the HPLC injection samples could be analyzed using a standardized HPLC procedure, each sample solution was filtered through a 0.2 μm membrane filter into a HPLC sample vial.

Instrumentation and conditions

The principal ingredients of all S. carvifolia extracts were analyzed by HPLC (Shimadzu, Japan) with PDA SPD M 20. A 20 μL sample loop was included with the LC-20 AD dual pump system and SIL-20 AC auto-injector (with cooler). Compounds were separated on a Shimadzu RP-C18 column with an internal diameter of 250 × 4.6 mm and a pore size of 5 m, which was protected by a guard column with the same packing. Subsequent elution of the column with hexane and a 2% ethyl acetate/hexane mixture yielded 100 mg of pure compounds infraction. The crude extract of leaf, stem, and roots was fractionated by 70% MeOH for 48 h before being injected three times with a 20 μL sample loop, and this ran for 25 min. Shimadzu Lab solution software was used to combine the data with the detection of peaks at 510 nm, and the findings were obtained by comparing them to accessible standards. For the detection of blank peaks, the plain mobile phase was employed as a control.

Total phenolic content

The total phenolic content (TPC) was determined from the leaf, stem, and roots of S. carvifolia extracts from different altitudes (2,150, 2,593, and 3,178 m) using the Folin–Ciocalteu method (52). Briefly, 1 mL of extract (100–500 μg/mL) solution was mixed with 2.5 mL of 10% (w/v) Folin–Ciocalteu reagent. After 5 min, 2.0 mL of Na₂CO₃ (75%) was subsequently added to the mixture and incubated at 50°C for 10 min with intermittent agitation. Afterwards, the sample was cooled, and the absorbance was measured utilizing a UV spectrophotometer (Shimazu, UV-1800) at 765 nm against a blank without extract. The outcome data were expressed as mg/g of gallic acid equivalents in milligrams per gram (mg GAE/g) of dry extract.

Total flavonoid content

The flavonoid contents of the leaf, stem, and roots of S. carvifolia extracts from different altitudes (2,150; 2,593; 3,178 m) were measured as per the aluminum chloride (AlCl3) assay (colorimetry). An aliquot of 1 mL of extract solution (25–200 μg/mL) or rutin (25–200 μg/mL) was mixed with 0.2 mL of 10% (w/v) AlCl₃ solution in methanol, 0.2 mL (1 M) potassium acetate, and 5.6 mL distilled water. The mixture was incubated for 30 min at room temperature followed by the measurement of absorbance at 415 nm against the blank. The outcome data were expressed as mg/g of quercetin equivalents in milligrams per gram (mg RE/g) of dry extract (53, 54).

Statistical analysis

The data were reported as the mean ± standard deviation. The linear regression coefficient (R²) for phenolic and flavonoid content with antioxidant activity was analyzed by Graph Pad Prism for Windows, Version 7 (Graph Pad Software, San Diego, CA, United States).

A linear regression analysis was used to obtain the IC50 values.

DPPH free radical scavenging assay

The free radical scavenging ability of the leaf stem and root extracts of S. carvifolia from different altitudes were tested by a DPPH radical scavenging assay (55). The hydrogen atom-donating ability of the plant extractives was determined by the decolorization of the methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH). DPPH produces violet/purple color in methanol solution and fades to shades of yellow color in the presence of antioxidants. A solution of 0.1 mM DPPH in methanol was prepared, and 2.4 mL of this solution was mixed with 1.6 mL of extract in methanol at different concentrations (12.5–150 μg/mL). The reaction mixture was vortexed thoroughly and left in the dark at RT for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. Ascorbic acid was used as a reference. The percentage DPPH radical scavenging activity was calculated by the following equation:

\begin{matrix} Free radical scavenging activity (%) = (A0) control - (A1) sample \\ / (A0) control * 100 \end{matrix}

where A0 is the control reaction absorbance, and A1 is the testing specimen absorbance. IC50 was determined graphically from the graph plotting the inhibition percentage (%) against the extracts (56).

Sugar-reducing capacity assay

The reducing sugar content (RSC) was determined using the 3,5-dinitrosalicylic acid (DNSA) method. The measurement was performed according to the procedure of Krivorotova and Sereikaite with slight modifications (57).

The DNSA reagent was prepared by dissolving 1 g of DNSA and 30 g of sodium–potassium tartaric acid in 80 mL of 0.5 N NaOH at 45° C. After dissolution, the solution was cooled down to room temperature and diluted to 100 mL with the help of distilled water. For the measurement, 2 mL of DNSA reagent was pipetted into a test tube containing 1 mL of plant extract (1 mg/mL) and kept at 95° C for 5 min. After cooling, 7 mL of distilled water was added to the solution, and the absorbance of the resulting solution was measured at 540 nm using a UV-VIS spectrophotometer (Shimadzu UV-1800). The reducing sugar content was calculated from the calibration curve of standard D-glucose (200–1,000 mg/L), and the results were expressed as mg D-glucose equivalent (GE) per gram of dry extract weight.

Antibacterial assay

Sample preparation and extraction

After cleaning, the different plant parts of the S. carvifolia plants collected from three altitudes (2,150, 2,593, and 3,178 m) were cut into small pieces using scissors and stored for drying. Drying was performed in a room for about 2 weeks in shade. They were then powdered using a grinder to enhance the surface area for a better extraction process. Twenty grams of finely ground powder from each plant part was used for making extracts. The 70% methanolic and aqueous extracts were prepared using a Soxhlet apparatus and simple maceration, and then the extracts were preserved for further studies.

Antibacterial activity

The antibacterial activity of the S. carvifolia leaf, stem, and root extracts from three different altitudes (2,150, 2,593, and 3,178 m) was evaluated against eight strains [3 Gram +ve Staphylococcus: Staphylococcus aureus (MTCC 96), Staphylococcus epidermidis (MTCC 435), and Streptococcus mutans (MTCC 890); 5 Gram –ve: Klebsiella pneumoniae (MTCC 109), Escherichia coli (MTCC 723), Escherichia coli (DH5α), Salmonella typhimurium (MTCC 98), and Pseudomonas aeruginosa (MTCC741); obtained from the Microbial Type Culture Collection Centre (MTCC), Institute of Microbial Technology (IMT), Chandigarh, India] by a micro-dilution broth assay using 96-well flat bottom microtiter plates according to the CLSI guidelines, according to which the antibiotic norfloxacin was used as a standard drug, and DMSO was used as a negative control (58).

Antifungal activity

By using the agar well diffusion method, the antifungal activity of all the S. carvifolia leaf, stem, and root extracts collected at different altitudes was investigated against four fungal strains: Candida albicans (CA-3010), Candida albicans (CA-227), Creptococcus neoformans (CN), and Sporothrix schenckii (SS) (59). All the fungal isolates were grown on a potato dextrose agar (PDA) at 28°C for proper sporulation. Subsequently, the fungal spores were harvested in sterile distilled water and evenly spread on PDA plates using a sterile glass spreader. Using a sterile cork borer (diameter 5 mm), wells were drilled into the agar media and filled with a sufficient amount of plant extract, oil, and water (control). The plates were allowed to rest at room temperature for 1 h to allow the extract to properly diffuse into the media before being incubated at 28°C for 96 h. The zones of inhibition around the wells were measured and recorded after incubation. The antifungal screening was performed in triplicates.

Cytotoxicity assay

Sample preparation and extraction

Selinum carvifolia leaves, stems, and roots were collected from three altitudes and air-dried for 10 days at room temperature before being milled into a powder. Following this, 70% methanol was used to extract the dry powder for roughly 7 days at room temperature. After evaporating the solvent, dry methanolic extracts were produced. To obtain suitable solutions for the extracts, dry methanolic extracts were dissolved in dimethyl sulfoxide (DMSO).

Cell culture

The study’s A549 cell line (adenocarcinoma human alveolar basal epithelial cells) was obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, United States). The cells were grown in Dulbecco’s modified Eagle medium (Sigma) supplemented with 10% FBS (fetal bovine albumin) and 1% antibiotic/antimycin cocktail at 37°C in humidified air containing 5% CO2. The cells were allowed to develop to 80–90% confluence before being harvested with 0.25% trypsin/EDTA solution, and they were sub-cultured in 96-well plates according to the experiment’s instructions.

Cytotoxicity assay

With minor changes (in concentration), the cytotoxicity assay based on MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) established by Mosman in 1983 was employed. A 5 mg/mL fresh stock solution of MTT was prepared in phosphate-buffered saline (PBS, pH 7.2) and filtered prior to initiating the experiment. Briefly, A549 cells (1 × 10⁴ cells/well) were seeded in a 96-well flat-bottom cell culture plate and cultured overnight (60). Furthermore, the cells were then treated with ethanolic extract of the plant at five different concentrations (1, 0.5, 0.25, 0.125, and 0.0625 mg/mL), as previously described (61). As a control, the cells were cultured in regular media (without extract and same culture conditions). After the treatment period was completed, 10 μL of MTT stock solution (1/10th volume of the total media in the well) was added to each well and incubated for 3 h under standard culture conditions. After the incubation period, the medium was withdrawn, and each experimental well was filled with 200 μL of DMSO (Sigma Aldrich), which was then incubated for 20 min at room temperature. Finally, MTT activity was measured using an ELISA plate reader set at 492 nm, and the absorbance intensity was recorded at a 550 mm wavelength (Biotek, PowerwaveXS2). The following formula was used to compute the percentage of growth inhibition:

% Cell inhibition = 100 - [(At - Ab) / (Ac - Ab)] \times 100

where At represents the absorbance value of the test substance, Ab represents the absorbance value of the blank, and Ac represents the absorbance value of the control.

IC50 values were used to express the effects of the extracts (the drug concentration reducing the absorbance of treated cells by 50% for untreated cells).

Results

HPLC analysis

To obtain an accurate quantification of chemicals, a sufficient resolution is required. The selection of the column, mobile phase composition, gradient flow parameters, and temperature was carried out by HPLC in order to obtain chromatograms with baseline separation of the 10 marker chemicals (ferulic acid, quercetin, gallic acid, rutin, chlorogenic acid, syringic acid, vanillic acid, cinnamic acid, caffeic acid, and protocatechuic acid) in S. carvifolia. To design a separation procedure for the isolates from the S. carvifolia extract solutions, a range of solvent systems were initially tested, starting with pure methanol and gradually adding the aqueous phase. The best-performing solvent system, consisting of methanol and water, was chosen for the current study. Eventually, all 10 phenolic marker chemicals were eluted in less than 40 min using a straightforward gradient approach based on water and methanol. With a fixed wavelength of 250 nm, the most effective detection was noted. The approach that was devised showed a good deal of specificity. The specificity of the developed analytical methods was determined by comparing the spectra’s peaks and retention durations. Consequently, we created a single stock solution in a standard manner before diluting it to the various concentration levels above and below the nominal amounts for each analyte. Based on their retention lengths and standard calibration curves, gallic acid, rutin, chlorogenic acid, syringic acid, vanillic acid, cinnamic acid, caffeic acid, and protocatechuic acid were identified and quantified from all plant components at all altitudes. In the herbal drug field, the standardization and characterization of herbal drugs are ongoing research interests together with formulations. With the development of contemporary chromatographic techniques, there is an increasing desire to create and develop simple, quick, practical, and affordable procedures for standardization. HPLC is a sensitive and precise tool that is frequently used for the quality assessment of plant extracts and the products that are made from them for standardizing the methanolic extract of plant leaves, stems, and roots (62, 63). The limitation of the study is that we have 10 phenolic compounds as markers, and these phenolic compounds have potent microbial and anticancerous activity; therefore, we only attempted to validate the plants that have these kinds of potent compounds that have been used for herbal drug formulations.

The HPLC fingerprints of S. carvifolia from three different altitudes (2,150 m, 2,593 m, 3,178 m) are given in Figures 1–3. The results of the HPLC analysis of S. carvifolia methanolic extract at 400 nm revealed eight different important chemicals found in various plant parts, as shown in Figure 1. Chlorogenic acid (0.25 mg/g) was identified in the smallest concentration in (PTE) stem extract, while gallic acid (13.93 mg/g) was discovered in the highest concentration in (Hx) stem extract in plants growing at an altitude of 3,178 m. Figure 2 summarizes the extract composition of different plant parts of S. carvifolia at an altitude of 2,593 m. The lowest concentration of chlorogenic acid (0.01 mg/gm) was discovered in the dry weight basis of (PTE) stem extract and (Hx) leaf extract, while the highest concentration of gallic acid (3.11 mg/gm) was discovered in the dry weight of (PTE) leaf. Similarly, at an altitude of 2,150 meters, Figure 3 summarizes the extract composition of plant parts of S. carvifolia. The results revealed that chlorogenic acid and syringic acid (0.01 mg/gm) were found in the lowest concentrations on dry weight in (PTE) and (Hx) leaf extracts, respectively, while chlorogenic acid (15.38 mg/gm) was found in the highest concentration in (PTE) stem extract and protocatechuic acid (15.12 mg/gm) in (PTE) stem extract. A single chromatogram of mixed standards and a single chromatogram of samples have been provided as Supplementary Data for reference.

Chemical composition of leaf, stem, and root extracts [(Chl), (Hx), and (PTE)] of *Selinum carvifolia* plants (in mg/gm) growing at an altitude of 3,178 m using HPLC showing the presence of chlorogenic acid, gallic acid, rutin, syringic acid, vanillic acid, cinnamic acid, caffeic acid, and protocatechuic acid.

Chemical composition of leaf, stem, and root extracts [(Chl), (Hx), and (PTE)] of *Selinum carvifolia* plants (in mg/gm) growing at an altitude of 2,150 m using HPLC showing the presence of chlorogenic acid, gallic acid, rutin, syringic acid, vanillic acid, cinnamic acid, caffeic acid, and protocatechuic acid.

Chemical composition of leaf, stem, and root extracts [(Chl), (Hx), and (PTE)] of *Selinum carvifolia* plants (in mg/gm) growing at an altitude of 2,593 m using HPLC showing the presence of chlorogenic acid, gallic acid, rutin, syringic acid, vanillic acid, cinnamic acid, caffeic acid, and protocatechuic acid.