Background
Tissues of many plants species contain secondary metabolites with the potential to combat disease-causing micro-organisms. These compounds include glycosides, saponins, flavonoids, steroids, tannins, alkaloids and terpenes [
1]. Extracts of different plant organs, including roots, leaves, bark, flowers, fruits and seeds, may contain distinct phytochemicals with activity against bacterial or fungal pathogens [
2]. In folk medicine, a single plant species is often used to treat more than one type of disease or infection [
3]. Extracts of plants with a history of traditional use should be tested using modern methods for activities against human pathogens, with the aim of discovering potential new drugs.
Inflammatory diseases comprising various kinds of rheumatism are common all over the world [
4]. Although rheumatism is the oldest known human disease, limited progress has been made for its permanent treatment. Non-steroidal anti-inflammatory drugs (NSAIDs) are being used to cure and control inflammation, fever and pain. However, their use has not been therapeutically efficacious in all types of inflammations. Furthermore, the use of NSAIDs can cause adverse side-effects leading to hemorrhage and ulcers [
5].
Pakistan has a rich diversity of medicinal and aromatic plants due to its unique phytogeography with diverse climatic conditions. The Chowk Azam town to the east of district Layyah lies in the sub-tropical continental plain of Pakistan, which features sandy soil. The climate is extremely hot in summer, while in winter temperatures can drop to 0 °C. People living in the villages around Chowk Azam rely mainly on medicinal plants to treat their minor diseases, and in some cases major diseases like cancer and hepatitis [
6].
Four medicinal plants were studied here:
Aristolochia indica (Aristolochiaceae), a creeper plant, perennial herb or shrub [
7];
Cuscuta pedicellata (Convolvulaceae), a holoparasitic annual that is usually observed as dense tangles of fine, yellow-orange, heavily-branched stems in the foliage of host plants [
8];
Melilotus indicus (Fabaceae), an annual noxious weed of crop fields, gardens, orchards and canal banks [
9] and
Tribulus terrestris (Zygophyllaceae), which features a stem that remains close to the ground, covered with lint [
10]. These plants are used by the local community in the villages of Chowk Azam in the district Layyah, Pakistan, for the treatment of various skin infections/irritations, poisonous bites, inflammation and some other infectious diseases (Table
1).
Table 1
Profile of ethnomedicinal use of selected medicinal plants, botanical/English/local name used, route of administration and plant extract yield obtained in the present study
1 |
Aristolochia indica L., Aristolochiaceae | Birthwort,wort killer/ Hukka-bel | Leaves | To cure poisonous bite, inflammations, reduce itching, leprocy, gastric stimulant, diarrhoea, intermittent fever, cough | Topical, Oral | 31 |
2 |
Cuscuta pedicellata Ledeb, Convolvulaceae | Clover dodder/ Loot booti (Saraiki) Akash-bail/Amar-bail (Urdu) | Stem | stomachache, to cure wounds, cuts, used as purgative, anti-inflammatory and to treat high blood pressure | Oral, Topical | 17 |
3 |
Melilotus indicus L., Fabaceae | Yellow melilot (English) Sweet clover/ Sinjee | Leaves | To cure inflammations and skin irritation, astringent, anticoagulant, laxative | Oral, Topical | 29 |
4 |
Tribulus terresteris L., Zygophyllaceae | Puncture clover/ Bhakra, Gokhru | Leaves | anti-inflammatory, lithotriptic, diuretic, general tonic | | 37 |
5 |
Tribulus terresteris
| | Fruit | anti-inflammatory, effective in most of Gynecological and genitourinary disorders, Gonorrhoea and to treat abdominal distension | Topical, Oral | 33 |
Key techniques employed in this study include the brine shrimp lethality and cytotoxicity assays, which are known to detect a wide range of bioactivities in plant crude extracts. The lethality potential of plant extracts to brine shrimp is an effective method of prescreening prior to conducting more elaborate antitumor and cytotoxicity assays. Numerous studies have reported the use of the brine shrimp assay to screen plant extracts for activities against fungi, arthropod pests (including insect larvae) and molluscs, as well as their anticancer and cytotoxic potential. The cytotoxicity brine shrimp assay has also been used successfully as a step in the identification of antineoplastic, cytotoxic, antimalarial, antifeedant and insecticidal compounds from many plants [
11].
The aim of this study was to determine whether the selected plant extracts can control the growth of pathogenic microorganisms. We focused on the phytochemical, antioxidant, antimicrobial, cytotoxicity and anti-inflammatory properties of the four medicinal plant species.
Methods
Chemicals and collection of plant materials
Methanol, Folin–Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), quercetin, rutin, gallic acid, aluminum chloride, potassium acetate, sodium acetate, ascorbic acid, hydrogen peroxide, phosphate buffer, nutrient broth, dimethyl sulfoxide (DMSO), potato dextrose agar, terbinafine, streptomycin, bovine serum albumin (BSA), casein, perchloric acid, aspirin, Tris-HCl, and trypsin were purchased from Sigma-Aldrich. All reagents and chemicals were of analytical grade.
Samples of the plant species
Aristolochia indica (AI)
, Cuscuta pedicellata (CP) (stems)
, Melilotus indicus (MI) and
Tribulus terresteris fruit (TTF) and leaf extracts (TTL) were collected from Chowk Azam, Layyah District, Punjab, Pakistan. The plants were identified by Prof. Dr. Mir Ajab Khan, Department of Botany, Quaid-i-Azam University. The plant species were selected on the basis of reports obtained from traditional herbalists and local people that extracts are used mainly to treat various infectious diseases. The scientific, English and local names, the parts of the plants extracted traditionally, their ethnomedicinal uses, route of administration and extract yields for the four plant species are described in Table
1.
Plant leaves, stems and fruits of the selected plant species were washed thoroughly with distilled water and shade-dried for 3 d at room temperature. The dried leaves, stems and fruits were uniformly ground using an electric grinder. The powdered plant material (250 g) was extracted for 4 d in 1 L 100% methanol [
12]. The separated extracts were then filtered through Whatman No. 1 filter paper and the methanol filtrate evaporated to dryness using a rotary evaporator at room temperature (30 °C). The thick extracted mass was then dried at room temperature, and the dried extract stored in an air-tight container at 4 °C until further use.
Yield percentage (
w/w) from the dried extracts was calculated as:
$$ \mathrm{Yield}\ \left(\%\right)=\left(\mathrm{W}1\ast 100\right)/\mathrm{W}2 $$
where W1 is the dry weight of extract after evaporating the solvent and W2 is the weight of the soaked plant powder.
Preliminary phytochemical screening
Phytochemical screening of the selected plant extracts was performed to detect the presence of phytochemical constituents, including saponins, terpenoids [
12], anthraquinones, phlobatannins [
13], flavonoids and phenolic compounds [
14].
Thin layer chromatography
The methanolic plant extracts (10 μL) were applied on pre-coated TLC plates using capillary tubes and air dried. The TLC plates were developed in a chamber using chloroform: methanol (5:1) as the mobile phase and observed under UV light (254 nm). Caffeic acid, quercetin, rutin, trans-cinnamic acid and salicylic acid were used as standards.
The mobility of the samples was expressed as retention factor (R
f) as calculated using the following formula:
$$ \mathrm{R} f=\frac{\mathrm{Distance}\ \mathrm{travelled}\ \mathrm{by}\ \mathrm{the}\ \mathrm{solute}\ \left(\mathrm{cm}\right)}{\mathrm{Distance}\ \mathrm{travelled}\ \mathrm{by}\ \mathrm{the}\ \mathrm{solvent}\ \left(\mathrm{cm}\right)} $$
Quantitative analysis of phytochemicals
Total phenolic content
Total phenolic content was analyzed using the Folin–Ciocalteu colorimetric method [
15] with some modifications. An aliquot of 0.3 mL of the plant extract was mixed with Folin-Ciocalteu phenol reagent (2.25 mL). After 5 min, 6% sodium carbonate (2.25 mL) was added and the mixture was allowed to stand at room temperature for 90 min. The absorbance of the mixture was measured at 725 nm in a spectrophotometer (HITACHI Model: U-1100 573 × 415). A calibration curve for gallic acid in the range 20–80 μg/mL was prepared in the same manner. Results were expressed as mg gallic acid equivalent (GAE) per gram extract.
Total flavonoid content
Total flavonoid content was determined using the aluminum chloride colorimetric method [
16,
17] with some modifications. A calibration curve for quercetin in the range 20–80 μg/mL was prepared. Plant extract (0.5 mL) and standard (0.5 mL) were placed in separate test tubes and 10% aluminum chloride (0.1 mL), 1 M potassium acetate (0.1 mL), 80% methanol (1.5 mL) and distilled water (2.8 mL) added and mixed. A blank was prepared in the same manner but 0.5 mL of distilled water was used instead of the sample or standard. All tubes were incubated at room temperature for 30 min and the absorbance was read at 415 nm. The concentration of flavonoid was expressed as mg quercetin equivalent (QE) per gram extract. Each plant extract was made in triplicate.
Total flavonol content
Total flavonol content was determined following the aluminum chloride colorimetric method [
18,
19] with some modifications. A calibration curve for quercetin in the range 20–80 μg/mL was prepared., Extract (1 mL) and standard (1 mL) were placed in separate test tubes and 2% aluminum chloride (1 mL), 5% sodium acetate (3 mL) added and mixed. The mixture was then centrifuged at 3000 rpm for 20 min to obtain a clear solution. The absorbance was read at 440 nm and the results expressed as mg quercetin equivalent (QE) per gram of extract. Each plant extract was prepared in triplicate.
DPPH radical scavenging activity
The free radical scavenging activity of the selected plant extracts was measured in vitro via the 2, 2′- diphenyl-1-picrylhydrazyl (DPPH) assay [
20,
21]. The reaction mixture (3 mL) consisted of 1 mL DPPH (0.3 mM) in methanol, 1 mL extract and 1 mL methanol. The radical scavenging activity of the samples at various concentrations (25–200 μg/mL) was measured. The reaction mixture was shaken well and incubated in the dark for 10 min at room temperature. Absorbance was read at 517 nm. The control was prepared as above but without any plant sample. Ascorbic acid [
22] and rutin [
23] were used as positive controls.
Scavenging activity was estimated based on the percentage of DPPH radical scavenged according to the following equation:
$$ \mathrm{Scavenging}\kern0.35em \mathrm{effect}\%=\left[\left(\mathrm{control}\kern0.35em \mathrm{absorbance}{\textstyle \hbox{--}}\mathrm{sample}\kern0.35em \mathrm{absorbance}\right)/\left(\mathrm{control}\kern0.35em \mathrm{absorbance}\right)\right]\times 100 $$
Hydrogen peroxide radical (H2O2) scavenging assay
The ability of plant extracts to scavenge hydrogen peroxide was determined according to the method of Ruch et al. (1989) [
24]. A solution of hydrogen peroxide (2 mM) was prepared in phosphate buffer (50 mM, pH 7.4). Plant extracts (25–200 μg powder/mL) were prepared in distilled water, and aliqots (0.1 mL) transferred into vials and their volumes made up to 0.4 mL with 50 mM phosphate buffer (pH 7.4). After addition of 0.6 mL hydrogen peroxide solution, tubes were vortexed and the absorbance was determined at 230 nm after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. Ascorbic acid [
25] and rutin were used as positive controls.
The ability of the extract to scavenge hydrogen peroxide was calculated using the following equation:
$$ \%\mathrm{Scavenged}\kern0.35em \left[{\mathrm{H}}_2{\mathrm{O}}_2\right]=\left[\left({\mathrm{A}}_{\mathrm{C}}{\textstyle \hbox{--} }{\mathrm{A}}_{\mathrm{S}}\right)/{\mathrm{A}}_{\mathrm{C}}\right]\times 100 $$
where A
C and A
S are the absorbance of the control and sample, respectively.
Pathogenic bacterial and fungal strains used
Bacteria
The bacterial pathogens used were Acinetobacter baumannii (ATCC 17978), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 7221) and Klebsiella pneumoneae (ATCC 6059), which were obtained from the Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan.
Fungi
The fungal pathogens Aspergillus flavus (FCBP-PTF-1265) and Aspergillus fumigatus (FCBP-MF-923) were obtained from the First Fungal Culture Bank of Pakistan (FCBP), University of the Punjab, Pakistan. Rhizopus oryzeae (ATCC 11886 (AY 803930)) was obtained from the Department of Microbiology, Quaid-i-Azam University, Islamabad.
The bacterial isolates were first sub-cultured in a nutrient broth (Sigma) and incubated at 37 °C for 18 h. The fungal isolates were sub-cultured on potato dextrose agar (PDA) (Merck) for 7 d at 25 °C.
Positive and negative controls
Streptomycin (30 μg/mL) and terbinafine (1 mg/mL) were used as positive controls for the antibacterial and antifungal tests, respectively. DMSO was used as negative control for the antibacterial and antifungal analyses.
Assay for antibacterial activity
Antibacterial activity of the methanol extracts of the selected plant species was determined using the agar well diffusion method [
26]. Petri plates were prepared by pouring 75 mL of seeded MH agar and allowing the agar to solidify. Freshly prepared bacterial inoculum was evenly spread using a sterile cotton swab on the entire agar surface. A hole was then punched with a sterile cork borer (6 mm) and 100 μL of each crude extract was poured into the well. Petri plates were then allowed to stand at room temperature for 1 h and incubated at 37 °C overnight. Controls were run in parallel whereby solvent was used to fill the well. The plates were observed for zones of inhibition after 24 h and the results compared with those of the positive control, streptomycin (30 μg/mL).
Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The determination of MIC of the methanolic extracts of the selected plant species was carried out by the microdilution method [
27] using nutrient broth. Plant extracts were dissolved in 10% DMSO and two-fold dilutions were prepared with culture broth. Each test sample and growth control (containing broth and DMSO, without plant extract/antimicrobial substance) was inoculated with 10 μL of bacterial suspension containing 5 × 10
6 CFU/mL. A 10-μL solution of resazurin (270 mg resazurin tablet dissolved in 40 mL of sterile water) was also added to each sample and incubated for 24 h at 37 °C. Bacterial growth was detected by reading absorbance at 500 nm. Bacterial growth was indicated by a color change from purple to pink or colorless (assessed visually). MIC was defined as the lowest plant extract concentration at which the color changed, or the highest dilution that completely inhibited bacterial growth. The test dilutions were further sub-cultured on fresh solid media and incubated for 18 h to determine the MBC values. The lowest plant extract concentration (highest dilution) that killed all the bacteria was defined as MBC. Experiments were carried out in triplicate to test each dilution for each bacterial strain to determine MIC and MBC values.
Assay for antifungal activity
The agar tube dilution method was used for the determination of antifungal activity of the methanol extracts of the selected plant species [
28]. Samples were prepared by dissolving crude plant extract in DMSO. Culture media was prepared by dissolving 6.5 g of potato dextrose agar per 100 mL distilled water (pH 5.6). Potato dextrose agar (10 mL) was dispensed in screw-capped tubes or cotton-plugged test tubes and autoclaved at 121 °C for 21 min. Tubes were allowed to cool at 50 °C and the potato dextrose agar was loaded with 67 μL of extract pipetted from the stock solutions. The tubes containing the media were then allowed to solidify in slanting position at room temperature. The tubes containing solidified media and plant extract were inoculated with a 4-mm-diameter piece of inoculum taken from a 7 d-old culture of fungus. Controls were run in parallel whereby the respective solvent was used instead of plant extract. The test tubes were incubated at 28 °C for 7 d. Cultures were examined twice weekly during the incubation. Readings were taken by measuring the linear length (mm) of fungus in the slant, and growth inhibition was calculated with reference to negative control. The experiments were performed in triplicate.
Percentage inhibition of fungal growth for each concentration of compound was determined as:
$$ \mathrm{Percentage}\ \mathrm{in}\mathrm{hibition}\ \mathrm{of}\ \mathrm{fungal}\ \mathrm{growth}=100-\frac{\mathrm{Linear}\ \mathrm{growth}\ \mathrm{in}\ \mathrm{test}\ \left(\mathrm{mm}\right)}{\mathrm{Linear}\ \mathrm{growth}\ \mathrm{in}\ \mathrm{control}\ \left(\mathrm{mm}\right)}\times 100 $$
Cytotoxic brine shrimp assay
Cytotoxic activity of the methanolic plant extracts was tested against brine shrimps hatched in saline solution (known as nauplii) [
29]. Methanol extracts of the selected plant species were prepared to make final concentrations of 10, 100 and 1000 mg/mL. An aliquot (2 mL) of each concentration was transferred to a graduated vial, kept for 48 h for solvent evaporation and then dissolved in DMSO before adding the nauplii. Brine shrimp eggs were hatched in a plastic rectangular container that was one-quarter filled with saline solution with general aeration. A plastic separator (with holes) for unequal compartmentation was placed in the container. Eggs were spread into the larger and darker compartment. After 48 h, mature nauplii were collected from the smaller and illuminated side. Ten shrimps were transferred to each vial and saline solution was added to make the final volume 2 mL. Vials were incubated at 25 °C for 24 h, after which the survivors were counted with the aid of a 3× magnifying glass. DMSO and saline solution were used as negative controls and potassium dichromate as the reference standard.
Lethal dose was calculated by linear regression analysis [
30].
Abbot’s formula was used to calculate the percentage mortality:
$$ \%\mathrm{Mortality}=\left(\mathrm{Sample}-\mathrm{control}/\mathrm{control}\right)\times 100 $$
Anti-inflammatory activity
Inhibition of protein denaturation method
Inhibition of protein denaturation was determined according to the method of Mizushima et al. [
31] with some modifications. The reaction mixture contained the test extract at different concentrations and 1% BSA (aqueous solution). 1 N HCl was used to adjust the pH of the reaction mixture. The samples were heated at 37 °C for 20 min and then 57 °C for 20 min, and allowed to cool. The turbidity of the samples was measured at 660 nm. The experiment was performed in triplicate. Percent inhibition of protein denaturation was calculated as follows:
$$ \mathrm{Percentage}\ \mathrm{inhibition}=\left({\mathrm{A}}_{\mathrm{C}}\ \mathrm{of}\ \mathrm{control}{\textstyle \hbox{--} }{\mathrm{A}}_{\mathrm{C}}\ \mathrm{of}\ \mathrm{test}\ \mathrm{sample}\right)\times 100/{\mathrm{A}}_{\mathrm{C}}\Big) $$
where A
C and A
S are the absorbance (at 600 nm) of the control and sample, respectively.
Human red blood cell (HRBC) membrane stabilization test
Fresh human blood (10 ml) was collected in heparinized centrifuge tubes and centrifuged at 3000 rpm for 10 min and washed 3× with an equal volume of normal saline solution. The volume of the blood was measured and reconstituted as a 10%
v/v suspension with normal saline [
32]. The reaction mixture (2 ml) consisted of 1 ml methanolic plant extract and 1 ml of 10% red blood cell suspension. For the control, saline was added instead of plant extract. Aspirin was used as a standard drug (positive control). The samples were incubated at 56 °C for 30 min, centrifuged at 2500 rpm for 5 min and the absorbance of the supernatant measured at 560 nm. The experiment was performed in triplicate. Percent membrane stabilization activity was calculated by the formula given in
Inhibition of protein denaturation method section [
33], while the percentage of protection was calculated using the following formula:
$$ \mathrm{Percent}\ \mathrm{of}\ \mathrm{protection}=100-{\mathrm{A}}_{\mathrm{S}}/{\mathrm{A}}_{\mathrm{C}}\times 100 $$
where A
C and A
S are the absorbance (at 560 nm) of the control and sample, respectively.
Proteinase inhibitory assay
The proteinase inhibitory assay was performed following the method modified by Oyedepo and Femurewa [
34]. The reaction mixture (2 ml) contained 0.06 mg trypsin, 1 ml Tris-HCl buffer (20 mM, pH 7.4) and 1 ml test plant extract sample at different concentrations. The reaction mixture was incubated at 37 °C for 5 min and then 1 ml of 0.8% (
w/
v) casein was added. The mixture was incubated for an additional 20 min. Perchloric acid (2 ml of 70%) was added to stop the reaction. The cloudy suspension was centrifuged and the absorbance of the supernatant was measured at 210 nm against Tris-HCl buffer as blank. The experiment was performed in triplicate.
Statistical analysis
Results were expressed as the mean ± standard error of mean (SEM). The data generated from quantitative assays for phytochemicals and antifungal activity were subjected to ANOVA using Statistix version 8.1. Comparison among mean values was made by Least Significant Difference (LSD) to test significant differences at
P < 0.05 [
35]. Linear regression analysis was used to calculate IC
50 values. Linear correlations were analyzed by using regression in R software (3.2.2.).
Discussion
The medicinal importance of Aristolochia indica (AI), Cuscuta pedicellata (CP), Melilotus indicus (MI) and Tribulus terrestris (TT) is well appreciated and reported from an ethnobotanical perspective but the biological activities of these plants collected from Chowk Azam, particularly CP, have been insufficiently investigated.
The selected plant extracts contain phenolic, flavonoid, tannin, terpenoid, phlobatannin and anthraquinone compounds. TLC profiling of all selected plant extracts in a chloroform: methanol solvent system also strongly suggested the presence of several bioactive metabolites in these plants. Furthermore, the quantitative analysis of tested plants revealed that methanolic stem extracts of CP
, TTF and TTL were rich in phenolics and flavonoids. According to Ali et al. [
36],
Cuscuta species contain alkaloids, some glycosides, tannins, flavonoids, steroids and phenolic compounds. We observed the maximum DPPH and H
2O
2 [
37] radical scavenging activities in the plant extracts of CP, TTF and TTL.
Total phenolic content is considered an important indicator of the antioxidant potential of plant extracts [
38]. The correlation coefficient between total phenolic content, DPPH and H
2O
2 scavenging activities found here suggests that the phenolic compounds of the selected plant extracts contributed 93% to their antioxidant activities. Similar results of positive correlations between phenolic content and antioxidant activities of several plant extracts have been documented in previous reports [
39,
40].
In the present study, the lowest MIC and MBC was observed in CP and found to be highly effective against the selected bacterial pathogens, followed by TTF > TTL extracts. Ali et al. [
36] and Faiyyaz et al. [
41] have also reported the antimicrobial potential of
C. pedicellata. The present study also revealed the antifungal potential of plant extracts against
A. flavus, A. fumigatus and
R. oryzae. Supporting this data, various medicinal plants are reported to show significant antifungal activity against
A. flavus [
42‐
44]. The antimicrobial potential of plant extracts can be attributed to the presence of certain bioactive compounds such as phenolics, tannins, flavonoids and polyphenols [
45]. Among all these biologically active compounds, Baydar et al. [
46] confirmed phenolics as the most significant and active compounds against bacteria as well as fungi. Similarly, the results of antibacterial and antifungal activities obtained in the present study were correlated to their total phenolic contents. Positive correlations between total phenolics of selected plant extracts and their antibacterial as well as antifungal potential were obtained.
Extracts are considered non-toxic if the LD
50 or LC
50 is greater than 100 μg/mL in the brine shrimp lethality assay [
47]. The mortality percentage and lethal dose (LD
50) for 50% of the population of nauplii were determined using statistical analysis and a graph of the logarithm of extract dose against lethality percentage [
48]. In the present study, the ranking order for cytotoxicity was TTF > TTL > AI > MI > CP. These results are supported by the findings of Menon et al. [
49] who reported the strong cytotoxicity of
T. terrestris fruit extracts. The anti-carcinogenic potential and antitumor activity of
T. terrestris fruit extracts [
47] have also been reported. Our results are also in accord with those of Hossen et al. [
50] who demonstrated the moderate cytotoxicity of
Aristolochia indica methanol extract. According to the previously reported literature, the compounds that show brine shrimp toxicity also tend to have cytotoxic properties against cells of solid tumors found in humans [
36].
Protein denaturation and stabilization of human red blood cell membranes were studied to further establish the mechanism of anti-inflammatory action of the traditionally used medicinal plants tested. Inflammation is usually associated with the denaturation of proteins. Results from the present study revealed that CP significantly inhibited protein/albumin denaturation. Methanolic stem extracts of CP had the highest anti-inflammatory potential (strong inhibition of protein denaturation), followed by the TTF and TTL extracts. The selected plant extracts were also effective in stabilizing RBC membranes or inhibiting the heat-induced hemolysis at different concentrations. Chowdhury et al. [
51] also reported that methanolic leaf extracts of
Gardenia coronaria promoted RBC membrane stability. Results from the present study provide evidence for membrane stabilization as an additional mechanism of their anti-inflammatory potential. The potential extracts (CP, TTF, TTL and MI) might inhibit the release of the lysosomal content of neutrophils at inflammation sites but this would need to be investigated. The lysosomal constituents of neutrophils include protease and bactericidal enzymes, which upon extracellular release cause more damage and tissue inflammation [
52].
Proteinases have been implicated in arthritic reactions. Neutrophils are reported to be a rich source of serine proteinases, which are localised in lysosomal granules. Leukocyte proteinases are involved in the development of tissue damage during inflammatory reactions and proteinase inhibitors provide substantial protection against this effect [
53]. Methanol extracts of
O. corniculata have been reported to have significant antiproteinase activity [
54]. In the present study, high correlation coefficients values were found between total phenolic content and anti-inflammatory as well as anti-proteinase inhibition activity of the selected plant extracts.
Conclusion
Maximum antioxidant, antimicrobial and anti-inflammatory activities were observed in methanolic CP extracts, which showed strong positive correlations with phenolic content. Results from this study revealed that CP stem extracts and TTF and TTL extracts contain a substantial phenolic content, which was suggested to be the major contributor to their antioxidant, antibacterial, antifungal, anti-inflammatory and anti-proteinase activities. These extracts have potential to be used to prevent food spoilage and to treat inflammation as well as skin irritations. Future research work will be focused on the use of CP stem extracts to protect against peroxidative damage related to carcinogenesis. The effectiveness of extracts of the medicinal plants studied here, particularly of CP, should be further elucidated through additional toxicity and phytochemical analyses to discover effective pharmacological agents.
Acknowledgements
The authors would like to thank the Higher Education Commission of Pakistan for providing funds to conduct this research.