Polarity guided extraction, HPLC based phytochemical quantification, and multimode biological evaluation of Otostegia limbata (Benth.) Boiss

2.5.2.1 Antileishmanial assay

The antileishmanial potential of test samples (extract) was assessed by MTT colorimetric assay according to previously documented procedure with minor changes (Ahmed et al., 2019). The Medium-199 (supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin sulphate, 100 IU/ml penicillin G) was used to culture Leishmania tropica kwh23 followed by incubation for 6–7 days at 24 °C. The promastigote culture (180 µl, seeding density of 1 × 10⁶ promastigotes/ml) was transferred to the respective well of 96 well plate already provided with test samples (20 µl). The test sample had not more than or equal to 1% DMSO in PBS with an absolute concentration of 100 µg/ml. Amphotericin B (0.33–0.004 µg/ml) was used as a standard while 1% DMSO in PBS served as a negative control. After incubation for 72 h at 24 °C, a stock solution of 4 mg MTT reagent per ml in distilled H₂O (20 µl, pre-filter, sterilized) was added to plate followed by another period of incubation (24 °C, 4 h). Next, the supernatant was isolated cautiously avoiding the disturbance of coloured formazan sediments. DMSO (100 µl) was mixed in well and allowed to set aside (1 h) to confirm the complete dissolution of formazan sediments. The absorbance was calculated via a microplate reader at 540 nm. The extracts with>50% cell mortality at 100 µg/ml were further studied at lower concentrations i.e., 33.3, 11.1, 3.7, and 1.23 µg/ml. IC₅₀ was evaluated using Graph pad prism 2D version 5.01 software.

2.5.2.2 Antibacterial assay

The agar disc diffusion method was employed to evaluate the antibacterial potential (In vitro) of test samples with minor amendments as described earlier (Khan et al., 2015). The refreshed bacterial cultures lawn: Staphylococcus aureus (ATCC-6538), Bacillus subtilis (ATCC-6633), Escherichia coli (ATCC-25922), Klebsiella pneumoniae (ATCC-1705), and Pseudomonas aeruginosa (ATCC- 15442) with pre-adjusted turbidity was developed on Petri plates imbedded with nutrient agar. The sterile filter paper discs infused with test extract (5 μl, 20 mg/ml DMSO) were directly placed on the surface of seeded plates. The cefixime and roxithromycin-infused disc (4 mg/ml in DMSO) worked as positive control whereas DMSO infused disc served as a negative control. After incubation (37 °C, 24 h), the average diameter of the inhibition zone adjacent to the discs was noted. The samples (extracts) showing an inhibition zone greater than or equal to 10 mm in diameter were considered as active followed by further screening to determine minimum inhibitory concentrations (MIC: lowest concentration of the extract inhibiting the growth of bacterial strain) by standard three-fold micro broth dilution method. The serial dilution method was employed on the stock solution of active samples in 96 well plate using Mueller Hinton broth to attain a range of concentrations (100, 33.33, 11.11, and 3.70 μg/ml). A standardized inoculum of every single bacterial strain (5 × 10⁴ CFU/ml) was transferred to the respective well. Following incubation (37 °C, overnight), MIC was assessed by calculating OD at 600 nm and the assay was completed as triplicate analysis.

2.5.2.3 Antifungal assay

The agar disc diffusion method was applied to assess the antifungal potential of test samples (extracts) in triplicate (Khan et al., 2015). The spores of given fungal strains [Aspergillus fumigatus (FCBP- 66), Mucor species (FCBP-0300), Aspergillus niger (FCBP-0198), and Aspergillus flavus (FCBP-0064)] were gathered in Tween 20 solution (0.02%). The turbidity of spores was adjusted conferring to McFarland 0.5 turbidity standard. Then harvested fungal strains (100 μl) were streaked on the plate’s surface having Sabouraud Dextrose agar. Sterile filter paper discs infused with test extract (5 μl, 20 mg/ml DMSO) were placed on the seeded plates. DMSO soaked disc acted as a negative control whereas standard drug clotrimazole served as a positive control. Following incubation (28 °C for 2448 h), the average diameter (mm) of the zone of inhibition around the disc was measured. Samples (extracts) showing inhibition zone ≥ 10 mm were further examined for MIC (minimum inhibitory concentration) estimation at lower concentrations (50, 25, 12.5, 6.25, and 3.12 μg/disc) by standard disc diffusion method.

2.5.4.1 Brine shrimp lethality assay

Brine shrimp lethality assay was carried out against Artemia salina (Brine shrimp) in 96 well plates for 24 h by the formerly adopted procedure with minor changes (Zahra et al., 2017). Eggs of Artemia salina (Ocean 90, USA) were allowed to hatch at 30–32 °C under direct light and constant provision of oxygen in a specially designed vessel with two compartments separated by a porous partition in sea water (38 g/l supplemented with 6 mg/l dried yeast) for 24–48 h. The mature nauplii were harvested with help of a Pasteur pipette and assembled in a small container. Next, the nauplii were transferred into 96 well plates containing test extracts (with serial dilutions), and volume was adjusted with sea water so that the concentration of DMSO was lower than 1%. The lethality of extracts was tested at four different concentrations i.e. 200, 100, 50, and 25 μg/ml. Doxorubicin (1.25–10 μg/ml) worked as standard, while DMSO was employed as a negative control. The percent lethality shown by each sample was measured by considering the number of alive nauplii after 24 h period. The test samples showing greater than or equal to 50% mortality were measured for median lethal concentration (LC₅₀) via Graph pad prism 2D version 5.01 software. The whole procedure was carried out three times.

2.5.4.2 Hemolytic assay

The hemolytic assay was carried out to assess the effect of various extracts from stem and leaf parts of O. limbata by previously documented procedure (Nasar et al., 2018). One ml of fresh human blood was taken in Eppendorf (1.5 ml) followed by centrifugation (14,000 rpm) to get RBCs (erythrocytes). After discarding the supernatant, an aliquot of 200 µl from blood (pellet) was kept in a falcon tube with the subsequent addition of phosphate buffer saline (PBS, 9.8 ml) and another cycle of centrifugation (10 min, 2000 rpm). The supernatant was thrown out and the washing process was repeated three times. A portion of 100 µl of red blood cell suspension (suspended in PBS) was transferred to a 96 well plate followed by the addition of stem and leaf extracts of O. limbata (dissolved in PBS). The plates were allowed to incubate for one hour (35 °C) followed by centrifugation at 1000 rpm for 10 min. The supernatant was collected and dispensed into 96 well plates. The release of hemoglobin was monitored by a microplate reader at 540 nm. Triton X-100 (0.5%) worked as a positive control, whereas PBS served as a negative control. Percent hemolysis was assessed using the following formula; $$\%\mathrm{H}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s}=(\mathrm{A}{\mathrm{b}}_{\mathrm{s}}-\mathrm{A}{\mathrm{b}}_{\mathrm{n}})/(\mathrm{A}{\mathrm{b}}_{\mathrm{p}}-\mathrm{A}{\mathrm{b}}_{\mathrm{n}})\ast 100$$

Whereas, Ab_s = Absorbance of the sample, Ab_n = Absorbance of negative control, and Ab_p = absorbance of positive control.

2.5.4.3 Cytotoxicity against THP-1 human leukemia cell line

Human leukemia (THP-1) cell line (ATCC # TIB-202) was used to assess the in vitro cytotoxic potential of test samples (crude extract) according to previously documented procedure with minor changes (Ahmed et al., 2019). Concisely, the complete growth medium comprising of RPMI-1640 buffered with 2.2 g/l NaHCO₃ and supplemented with 10% HIFBS (heat-inactivated fetal bovine serum) was used to culture leukemia cells at 37 °C (7.4 pH) in a 5% humidified CO₂ incubator (Panasonic, Japan MCO-18 AC-PE) for 72 h. A portion of 190 μl of THP-1 cells was transferred to the respective well of 96 well plate with a defined seeding density (5 × 10⁵ cells per ml). Afterward, the sample (10 μl) having not>1% DMSO in phosphate buffer saline was added. Samples were evaluated at a concentration of 20 μg/ml three times. The reaction mixture was allowed to incubate in a humidified CO₂ incubator with 5% CO₂ at 37 °C for 72 h. The standard drugs used were vincristine and 5-florouracil prepared at a final concentration of 20 μg/ml. 1% DMSO solution in PBS served as a negative control. An amount of 20 µl of MTT solution (4 mg/ml in distilled water, sterile filtered) was infused in plate followed by incubation in a CO₂ incubator (5%) at 37 °C for 4 h. Afterward, the formazan sediments were carefully removed. DMSO (100 µl) was mixed in the respective well and set aside for 1 h to make sure the complete dissolution of formazan sediments. Eventually, the absorbance was noted via a microplate reader at 540 nm. Samples presenting ≥ 50% inhibition were further examined at lesser concentrations of 6.66, 2.22, 0.74, and 0.24 μg/ml. The analysis was carried out three times. The inhibitory concentration (IC₅₀) was measured through Graph pad prism 2D version 5.01 software.

2.5.4.4 Cytotoxicity against Hep-G2 cell line

Cytotoxic potential of the samples was assessed via SRB colorimetric assay against Hep-G2 cell line (RBRC-RCB1648) according to a previously documented process with minor changes (Ahmed et al., 2019). DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 100 μg/ml streptomycin sulphate, 10% FBS, 100 IU/ml penicillin G sodium and 0.25 μg/ml amphotericin B was employed for the proliferation of Hep-G2 cells. The plate was allowed to incubate in CO₂ humidified incubator (5% CO₂) at 37 °C for 72 h to achieve 70–80% confluence of cells. The fresh medium was used and another 24 h incubation period was subjected to cells followed by trypsonisation and dilution to achieve the required seeding density (1 × 10⁵ cells/ml). The test sample (20 μl) containing 1% DMSO in PBS was added to a specific well of 96 well plate. Afterward, an aliquot of 180 μl is added to each well from Hep-G2 culture to achieve a final concentration of 20 μg/ml. Doxorubicin with concentrations 20, 6.66, 2.22, 0.74, 0.24, and 0.08 μg/ml in Phosphate Buffer Saline served as positive control whereas 1% dimethyl sulfoxide in PBS worked as a negative control. After another period of humidified incubation (37 °C for 72 h in a CO₂ incubator), the culture plate was supplied with 50 μl of cold TCA solution for cell fixation (20% w/v) followed by washing with tap water (4 times). Subsequently, the plate was dried and stained with SRB reagent (50 μl of 0.057% w/v SRB in 1% w/v acetic acid) for 30 min at room temperature. After a series of repeated washing with acetic acid (1% v/v), plates were allowed to dry overnight. An amount of 200 μl of Tris base (10 mM) at pH 10 was used to dissolve the bound dye and maintained at room temperature for 1 h. A microplate reader (Biotech USA, microplate reader Elx 800) was used to measure the optical density at 515 nm and percent survival of cells was noted. Zero-day control was run by the addition of cells in 16 wells in equal amount for 1 h at 37 °C and the same procedure was followed as described earlier. Percent inhibition was measured by the following formula: $$\%\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=100-\frac{\mathrm{O}{\mathrm{D}}_{\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}+\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{s}}-\mathrm{O}{\mathrm{D}}_{\mathrm{d}\mathrm{a}\mathrm{y}0}}{\mathrm{O}{\mathrm{D}}_{\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}+1\%\mathrm{D}\mathrm{M}\mathrm{S}}-\mathrm{O}{\mathrm{D}}_{\mathrm{d}\mathrm{a}\mathrm{y}0}}\times 100$$

2.5.4.5 Protein kinase inhibition assay

The protein kinase inhibition assay was accomplished by using a purified strain of Streptomyces 85E (Zahra et al., 2017). The tryptone soya broth medium was utilized to refresh the strain of Streptomyces 85E for 96 h at 37 °C. The mycelia fragments (spores) of Streptomyces were dispersed in minimal ISP4 medium on sterilized plates to grow the bacterial lawn. An amount of 5 μl from each sample stock solution (20 mg extract /ml of DMSO) was loaded onto a sterile filter paper disc (6 mm). The saturated paper discs having an absolute concentration (100 μg/disc) were placed on the surface of plates imbedded with Streptomyces 85E. Surfactin acted as positive control while DMSO served as a negative control. The plates were incubated approximately for 72 h (time necessary for hyphae formation) at 30 °C. The results were inferred as a bald zone of inhibition around the discs.

3.2.2.1 Antileishmanial activity

Antiproliferative activity against Leishmanial promastigotes based on MTT assay is summarised in Fig. 3. Among the stem extracts, noteworthy activity was observed in Hex, CHCl3, EtOAc-Hex, and MeOH-EtOAc extracts (IC₅₀ values ranging from 4.45 ± 0.45 to 11.67 ± 0.45 μg/ml) while MeOH-CHCl3, ACET, and D.W.-ACET extracts showed moderate activity (IC₅₀ = 21.75 ± 0.45 μg/ml). In the case of leaf extracts, remarkable inhibition was recorded in MeOH-D.W., MeOH, EtOH, and MeOH-CHCl3 extracts with IC₅₀ of 1.50 ± 0.23, 4.66 ± 0.23, 5.78 ± 0.20, and 11.11 ± 0.01 μg/ml whereas EtOAc-Hex, EtOH-Hex, ACET, MeOH-ACET, D.W.-ACET and D.W. samples showed moderate activities (IC₅₀ = 100 ± 0.45 μg/ml). The positive control (amphotericin B) showed an IC₅₀ value of 0.01 μg/ml while DMSO (negative control) did not show any activity.

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Fig. 3

The antileishmanial potential of O. limbata extracts. Amphotericin B (positive control) showed IC₅₀ value of 0.01 μg/ml. Values are presented as mean ± standard deviation from the triplicate investigation. The columns with different superscript (^a-d) letters show significantly (P < 0.05) different means.

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3.2.2.2 Antibacterial activity

The antibacterial potential of O. limbata extracts against various bacterial strains is documented in Table 2. Most of the extracts were found to be active with a minimum zone of inhibition ≥ 10 mm. A prominent zone was observed against P. aeruginosa by EtOH extract (13 ± 0.56 mm, MIC = 33.3 ± 0.52 μg/ml) followed by 71% stem extracts with zone ranging from 10 to 12 mm at a concentration of 100 μg/disc. The Hex extract of stem displayed the best inhibitory potential against B. subtilis with 16 ± 0.44 mm diameter (MIC = 33.3 ± 0.53 μg/ml) followed by 35% extracts (ZOI = 10–12 mm). A noticeable zone of inhibition was detected against S. aureus by various stem extracts including D.W.-ACET and MeOH-D.W. (MIC = 33.33 ± 0.37 μg/ml) extracts. Several leaf extracts have shown noteworthy antibacterial affect against E. coli, P. aeruginosa, S. aureus, B. subtilis, and K. proteus. In the case of leaf extracts, the most significant activity was documented against P. aeruginosa by EtOH extract (MIC = 33.3 ± 0.55 μg/ml) followed by EtOH-Hex, MeOH-CHCl3, CHCl3, and MeOH-D.W. extracts with MIC value of 33.3 ± 0.46 μg/ml. Noticeable activity against B. subtilis was recorded by MeOH-ACET and ACET extracts (MIC = 33.3 ± 0.56 μg/ml) whereas CHCl3 extract showed potent activity against S. aureus (MIC = 33.3 ± 0.65 μg/ml) in leaf extracts. The zones produced by the extracts were comparable to the standard cefixime which produced 18 ± 0.45 mm zone of inhibition at 10 μg/disc concentration. The activity was more intensified in non-polar and moderately polar extracts. The negative control (DMSO) showed no inhibition.

3.2.2.3 Antifungal assay

The results of antifungal propensity against four kinds of fungal strains are depicted in Table 3. In the case of stem extracts, CHCl3 and ACET exhibited most prominent antifungal activities against A. fumigatus, Mucor sp, A. flavus, and A. niger with MIC values ranging from 6.25 to 50 µg/disc respectively. The most significant activity against A. flavus waspresented by 85% stem extracts wherein EtOAc-Hex extract showed the best activity with a MIC value of 6.25 µg/disc. Hex, CHCl3, EtOAc, ACET, and EtOH extracts also presented good antifungal potential against the Mucor species with MIC = 6.25 µg/disc. In the case of leaf extracts, the most prominent antifungal potential was presented against A. niger by Hex, CHCl3 and EtOAc extract with the MIC value of 12.5 µg/disc. The noticeable activity against A. fumigatus was revealed by 42% leaf extracts whereas the most potent activities were documented by EtOH extract (MIC = 6.25 µg/disc). Substantial antifungal action was also exhibited against Mucor species by several leaf extracts. Clotrimazole as a positive control revealed the maximum antifungal propensity however DMSO did not show any antifungal tendency.

3.2.4.1 Brine shrimp lethality test

Preliminary cytotoxicity was assessed by brine shrimp lethality assay and results have been presented in Table 4. Among the leaf extracts, 57% of the extracts (8 out of 14 extracts) showed moderate toxicity with LC₅₀ values of 50–200 μg/ml while Hex, MeOH-CHCl3, MeOH, CHCl3 and ACET leaf extracts were most potent (LC₅₀ = 50–59 μg/ml). In the stem extracts, 21% extracts (3 out of 14 extracts) were found to be moderately cytotoxic (LC₅₀ = 70–83 μg/ml) while the remaining extracts were weakly cytotoxic. The positive control, doxorubicin revealed LC₅₀ value of 5.63 ± 0.21 µg/ml while the negative control (≤1% DMSO in sea water) did not show any toxicity.

3.2.4.2 Hemolytic assay

The results of hemolytic activity of extracts estimated against freshly isolated erythrocytes (human RBCs) are presented in the Table 4. Both stem and leaf extracts presented very low percent hemolysis at concentrations of 200 μg/ ml. This model was used to evaluate the hemolytic activity of extracts to normal body cells (RBCs).

3.2.4.3 Cytotoxicity against THP-I cell line

All extracts were tested at 20 μg/ml against leukemia (THP-1) and samples presenting inhibition>50% were further evaluated at lower concentrations (6.66, 2.22, 0.74 μg/ml) to determine the IC₅₀ as given in Table 4. The leading cytotoxic activity was displayed by MeOH-EtOAc, Hex, MeOH-ACET, CHCl3, and ACET with IC₅₀ of 5.9 ± 0.43, 6.6 ± 0.43, 7.01 ± 0.4, 19.3 ± 0.4, and 20 ± 0.45 μg/ml regarding stem extracts. A remarkable cytotoxic potential was displayed by leaf extracts including MeOH-D.W., MeOH, MeOH-CHCl3, EtOH, MeOH-ACET, ACET, and Hex with IC₅₀ values of 3.46 ± 0.25, 3.98 ± 0.42, 4.58 ± 0.43, 5.67 ± 0.36, 8.43 ± 0.32, 9.01 ± 0.43, and 20 ± 0.51 μg/ml respectively greatly comparable to standard drugs. The standard drugs employed were 5-florouracil and vincristine having IC₅₀ of 5.13 ± 0.21 and 8.24 ± 0.22 μg/ml respectively.

3.2.4.4 Cytotoxic potential against Hep-G2 cell line

The results of cytotoxic potential of O. limbata extracts against Hep-G2 cell line have been mentioned in Table 4. Among stem extracts, the most promising activity was found in Hex, CHCl3, ACET, MeOH-ACET, MeOH-EtOAc, and MeOH-CHCl3 with IC₅₀ values of 0.44 ± 0.45, 1.1 ± 0.56, 1.86 ± 0.36, 1.98 ± 0.52, 2.02 ± 0.45, and 3.24 ± 0.33 μg/ml respectively even superior to standard doxorubicin (IC₅₀ = 5.30 ± 0.67 μg/ml) while other extracts (57.14%) showed moderate activity. In the case of leaf extracts, most powerful activities were shown by; MeOH-D.W., Hex, EtOAc, MeOH-CHCl3, MeOH-ACET, EtOH, and MeOH extracts with IC₅₀ values of 1.33 ± 0.43, 1.44 ± 0.65, 2.19 ± 0.54, 2.56 ± 0.66, 3.26 ± 0.33, 3.46 ± 0.37, and 3.55 ± 0.67 μg/ml respectively whereas remaining 50% of extracts exhibited a moderate Hep-G2 inhibition with IC₅₀ values>20 μg/ml. Standard drug (doxorubicin) unveiled an IC₅₀ of 5.30 ± 0.67 μg/ml

3.2.4.5 Protein kinase inhibition potential

After a sound in vitro anticancer study, the extracts were further screened for protein kinase inhibitory potential with results arranged in Table 4. In the case of stem extracts, a noteworthy bald zone of growth inhibition was observed around MeOH-D.W. (26 mm) and D.W. (24 mm) samples with IC₅₀ = 12.5 μg/disc followed by descending order of MeOH-ACET > Hex = MeOH > EtOH > MeOH-CHCl3 extracts. In the case of leaf extracts, noticeable hyphae formation inhibition (bald) zones were shown by MeOH, EtOAc-Hex, CHCl3, and Hex extracts impregnated discs with an IC₅₀ value of 12.5 μg/disc. The rest of the active samples showed prominent bald zones with the following decreasing order; MeOH-EtOAc > ACET > EtOH-Hex > D.W. = MeOH-CHCl3. As a negative control (DMSO) did not exhibit a bald zone whereas surfactin (positive control) showed a prominent bald zone (28 mm).

4.2.2.1 Antileishmanial potential

Leishmaniasis is a widespread disease universally affecting 12 million people in 88 countries with almost 350 million individuals threatened. There are various species of Leishmania responsible for visceral, cutaneous, and mucocutaneous leishmaniasis (Bhunia and Shit, 2020). Leishmania tropica is a flagellated parasite causing cutaneous leishmaniasis (Masoudzadeh et al., 2020). Pentavalent antimonials are standard drugs for the treatment of leishmaniasis nevertheless leading to renal toxicity (Marques et al., 2019). The emergence of resistance to antimonials, some serious side effects, and incidence of disease worldwide emphasizes the discovery of leishmanicidal agents (Mambro et al., 2019).

To the best of our knowledge, this study discloses the potential against L. tropica for the first time. Plants constitute a different kind of phytoconstituents such as alkaloids, terpenoids, flavonoids, and tannins as a source of antioxidants in many studies. The lack of certain enzymes such as GSH peroxidases and catalases render the parasite more vulnerable to reactive oxygen species (ROS) mediated programmed cell death (apoptosis). Flavonoids such as quercetin show antileishmanial action by inhibition of topoisomerase II causing cell cycle arrest (Ahmad et al., 2020). Some former studies reported that clerodane diterpenoids revealed 86.9%, 83.7%, and 84.2% inhibition of L. donovani amastigotes in spleen, liver, and bone marrow respectively at 100 mg/kg dose in hamsters (Duarte et al., 2019). Farooq et al (2004) revealed the presence of some clerodane type diterpenoids such as limbatolide and limbatenolide in methanol extract of O. limbata providing evidence of leishmanicidal action in the current study. Therefore, the incidence of flavonoids confirmed by HPLC in the current study along with diterpenoids reported in earlier studies may be attributed to the antileishmanial potential of O. limbata. Overview of whole data led us to the conclusion that stem contains most active compounds in non-polar region and leaves have bioactive compounds concentrated in the polar region. Therefore, this information could be further employed to isolate and characterize pure compounds with the potential to develop antileishmanial drugs in the future.

4.2.2.2 Antibacterial potential

The manifestation of antibacterial action highly depends upon the plant material utilized, methods applied for testing, growth medium composition, and the strains used (Vieitez et al., 2018). The extracts are specifically active against B. subtilis strain thus qualifying as a valuable source of narrow-spectrum antibiotics. Monotherapy with narrow-spectrum drugs provides the advantage of a targeted approach which is considered valuable to avoid the emergence of resistance widely seen in the case of broad-spectrum antibiotics (Khameneh et al., 2016). Moreover, phytochemical analysis confirmed the presence of rutin, caffeic acid, apigenin, and gallic acid in the current study. A significant antibacterial potential is reported by rutin and apigenin (Saleh et al., 2019; Sen et al., 2016). Inhibition of certain bacteria including P.aurogenosa, S.aureus, and Streptococcus mutants by gallic acid has also been reported. Mechanism of inhibition involves hindrance in motility, adherence as well as biofilm formation. Gallic acid may also affect the permeability and charge of the membrane. Some studies have reported the effect of gallic acid on some metabolic enzymes including dihydrofolate reductase and topoisomerase IV leading to DNA cleavage. Chelation via divalent cations may also contribute to membrane disruption of gram-negative bacteria (Kahkeshani et al., 2019).

Previous studies have reported the role of methanol extract and the fraction of the whole O. limbata plant in antibacterial proficiency (Shakoor and Zaib, 2014). Other studies also showed the presence of diterpenoids (Ahmad et al., 2005). It can be deduced that this plant is rich in the aforementioned chemicals as confirmed by the phytochemical analysis and it could be a source of natural compounds to develop new antiinfective agents.

4.2.2.3 Antifungal potential

The current data suggests that the most substantial antifungal action (MIC = 6.25–50 μg/ml) is prominent in the stem part compared to the leaf part. Flavonoids (Caffeic acid, apigenin, rutin, and gallic acid) occurrence in several extracts of both stem and leaf parts of O. limbata has been confirmed by chromatographic fingerprinting (RP-HPLC) in the current study. Flavonoids are widely used to treat several human ailments. The antifungal mechanism of flavonoid involves several fundamental principles such as inhibition of cell membrane and cell wall formation, inhibition of RNA and protein synthesis, dysfunction of the mitochondrial system, and efflux pumps (Aboody and Mickymaray, 2020). To the best of our knowledge, the current study reveals the antifungal activity against A. flavus and Mucor species for the first time. The potent antifungal action of the subject plant needs to be further investigated for isolation of bioactive to treat fungal infections.

4.2.3.1 Several methods

Clerodane type diterpenoids have been documented in O. limbata extracts in some earlier reports (Ahmad et al., 2007). Previous studies have described the role of clerodane diterpenoids in the inhibition of α-amylase (Huang et al., 2019; Luyen et al., 2019). This enzyme catalyzes the hydrolysis of glycosidic linkages (α 1–4) from the non-reducing region of polysaccharides like amylopectin, starch, and glycogen to produce maltose leading to increased glucose absorption. The α-amylase inhibition decreases glucose absorption. Hence postprandial glucose upsurge is reduced (Bashary et al., 2020). Polyphenols and flavonoids act as an antioxidant, decrease oxidative stress, obstruct the free radicals production, and inhibit digestive enzymes hence reducing postprandial glucose rise (Gutiérrez-Grijalva et al., 2019). The current study reveals the diabetic-friendly potential of O. limbata for the first time that should be further investigated via purification and characterization to develop more safe targets for this chronic disease.

4.2.4.1 Brine shrimp lethality

These results suggest that a non-polar and moderately polar binary solvent system is the best option for cytotoxic compound isolation as compared to a most polar solvent systems in O. limbata stem and leaf extracts. Previous studies reported that the whole plant of O. limbata was evaluated for cytotoxic potential on rhabdomyosarcoma (RB) cell line using methanolic extract and it exhibited anticancer activity emphasizing the cytotoxic potential of the plant (Maqsood et al., 2015). The brine shrimp lethality assay is a well-planned method for primary assessment of toxicity (Hamidi et al., 2014). The current study revealed the brine shrimp lethality potential by various parts of O. limbata for the first time. The LC₅₀ value below 1000 μg/ml is considered cytotoxic during bioactivity evaluation via brine shrimp assay in plant extracts. Therefore, the cytotoxic potential was further assessed by hemolytic assay, cancer cell lines (THP-1 and Hep-G2), and protein kinase inhibition assays.

4.2.4.2 Hemolytic activity

Hemolytic potential illustrated the mechanism of cytotoxicity to cell membrane since RBCs membrane resembles other cell membranes (Alonso and Alonso, 2016). Both stem and leaf extracts displayed very low percent hemolysis indicating the biocompatibility with normal body cells. Negligible hemolysis at a concentration of 200 μg/ ml represents the selective cytotoxicity of extracts towards the THP-1 and Hep-G2 cancer cell line in comparison to normal body cells (RBCs). The entire data of hemolytic assay led us to the conclusion that stem and leaf extracts of O. limbata have notable biocompatibility and do not distress the normal body cells. To the best of our knowledge, this is the first-ever report regarding the hemolytic potential of O. limbata. The hemolytic activity should be further investigated to use these extracts for food and drug products.

4.2.4.3 Cytotoxicity against THP-1 cell line

The stem extracts of O. limbata showed more cytotoxic potential in non-polar and moderately polar binary solvents. The leaf extracts showed more percent inhibition against THP-1 cell line in moderately polar binary solvents to polar solvents which can be correlated to a previous study on methanol extract of O. limbata that proved effective against rhabdomyosarcoma cell line (Maqsood et al., 2015). Anti-cancer properties of rutin (flavonol) were observed by in vitro and in vivo studies (Saleh et al., 2019). Apigenin as a free radical scavenger possesses antibacterial, anti-proliferative, and anti-inflammatory effects (Sen et al., 2016). Several in vitro and in vivo studies illustrated treatment prospects of gallic acid regarding ulcer, colorectal cancer, and diabetes (Kahkeshani et al., 2019). Quantification of these phytochemicals (Rutin, apigenin, and gallic acid) based on RP-HPLC analysis along with clerodane type diterpenoids such as limbatolide and limbatenolide reported by Farooq et al. (2007) may be attributed to the antileukemic potential of tested samples. Vincristine employed as a positive control in the current study has been widely used in several categories of cancer including acute lymphoblastic leukemia, Hodgkin lymphoma, Wilms’ tumor, sarcomas, and neuroblastoma. Vincristine-induced hemolytic anemia is extensively reported in clinical settings (Michlitsch et al., 2019). The tested extracts of O. limbata revealed negligible percent hemolysis at a concentration of 200 μg/ml. Accordingly, the results validate that samples from both stem and leaf extracts have greater selectivity for THP-1 cell line compared to normal body cells (RBCs).

This is the first report describing the anticancer potential against monocytic leukemia. This suggests that a high degree of selective cytotoxicity of stem and leaf extracts of O. limbata against human monocytic leukemia should be employed further for purification and characterization of the cytotoxic compounds from potent extracts use candidates for chemotherapeutic drug development.

4.2.4.4 Cytotoxicity against Hep G2 cell line

The greater cytotoxic potential was documented by several extracts in comparison to standard drugs employed in the current study. The data shows that the cytotoxic profile of the stem extracts mainly resides in the non-polar region which convinces the non-polar nature of active moieties. The presence of clerodane type diterpenoids in previous reports may be correlated to the anticancer effect of Hex stem extract against cancer cell lines in the current study. Similarly, clerodane type diterpenoids isolated from Scutellaria barbata imparts anticancer effects as observed by in vitro studies against various human carcinoma cells, including, breast cancer, colon cancer, and hepatocellular carcinoma by cell cycle arrest, apoptosis, and autophagic pathways (Wang et al., 2019). Therefore, literature also supports the anticancer potential of non-polar extracts clerodane diterpenoids. Among leaf extracts, polar and moderately polar extracts reported greater cytotoxic potential. Anticancer properties of rutin, gallic acid, and apigenin regarding ulcer, colorectal cancer, and diabetes were described in earlier studies (Kahkeshani et al., 2019; Saleh et al., 2019; Sen et al., 2016). Significant quantification of these phytochemicals based on HPLC in the present study accentuates the cytotoxic potential of polar and moderately polar extracts of O. limbata.

The results also validate that the samples from both stem and leaf extracts have greater selectivity for Hep-G2 cell line compared to normal body cells (RBCs). This suggests that a high degree of selective cytotoxicity of stem and leaf extracts against Hep-G2 hepatoma cell line should be employed further for purification and characterization of cytotoxic compounds from potent extracts for chemotherapeutic appraisal.

4.2.4.5 Protein kinase inhibition potential

Several extracts of both stem and leaf parts exhibit notable inhibitory activities. The data exhibited that polar solvents would be a better choice for the extraction of active constituents from the stem part while non-polar and moderately polar solvents would efficiently extract the phytochemicals from the leaf part of O. limbata with kinase inhibitory activity.

Cancer pathogenesis involves the accumulation of mutations in the genes crucial for the cellular differentiation, growth, and apoptosis. The mutations constitutively upraise the activity of kinase at serine/threonine residues, frequently observed in human cancers. The serine/threonine kinases are an important factor in carcinogenesis (Roskoski, 2019). Similarly a subfamily of serine/threonine kinases was mutated in ovarian carcinomas (Bhullar et al., 2018). The tyrosine kinases control the normal cell functioning and also play a main role in oncogenesis (Qu et al., 2020). All these kinases are reflected as exceptional targets for cell signaling involved in the cancer pathogenesis as well as to limitize the toxicities exhibited by conventional therapy (Montagnani and Stecca, 2019). The protein kinase inhibitory potential could be determined by use of Streptomyces 85E strain. This strain closely looks like the eukaryotic counterpart. The inhibition this strain by several extracts helps to recognize the anti-infective, antitumor agents and antimycobacterial potential of plant (Belknap et al., 2020; Zahra et al., 2017). Further investigations are required to exploit these bioactivities of the plant.