Extracts preparation

Fresh leaves of five selected species were collected and their accession numbers were assigned from the Herbarium of the National Agriculture Research Centre (NARC), Islamabad. The extracts were weighed to determine the extract yield (Table 1).

Determination of phytochemicals

For total phenolic contents (TPC), total flavonoid contents (TFC) and total alkaloid contents (TAC), the procedure was the same as the previously reported method(11,12, 13).

Antioxidant assay

In this assay, 0.1 mM DPPH solution was made by adding 3.94 mg of DPPH in 100 mL of methanol, and the absorbance (0.98 ± 0.02) was set at 517 nm. The procedure was followed as per the previously reported method¹⁴.

DNA damage protection assay

Briefly, 14 mg of iron sulphate heptahydrate (FeSO4.7H2O) was added to 25 ml of distilled water (dH2O), and 0.2 µg/µl pBR322 plasmid was diluted using 50 mM of phosphate buffer saline (PBS). Then, 3 µl of pBR322 plasmid DNA was added in Fenton reagent and plant extracts, and the volume was made up to 15 µl using dH2O. The reaction mixture was incubated (30 min) at 37 °C, and then 1 % agarose gel was prepared. Subsequently, 5 µl of ethidium bromide (EtBr) was added to the gel and transferred to the gel tank, using 1x Tris-borate EDTA (TBE) as a running buffer. The reaction mixtures (10 µl) were loaded on the gel, and 2 µL of loading dye and electrophoresis were conducted. After 20-30 min, DNA bands were observed under UV light¹⁵.

Antidiabetic assays

Anti-leishmanial assay

For parasites culture, L. major isolates were obtained from the Department of Zoology, University of Peshawar (Pakistan) and grown in RPMI-1640 culture medium with 10 % heat-inactivated fetal calf serum (HIFCS) in the presence of penicillin and streptomycin solution (100 µg/mL each) at 23 °C. The MTT (3-(4,5-dimethylthiazole-2yl)-2,5-diphenyltetrazolium bromide) assay was done per the previously reported protocol¹⁸.

Anti-dengue assay

A. aegypti larvae were collected with an aquatic net and then transferred to the laboratory. The larvae were fed dog biscuits and yeast powder (3:1) and maintained at 28 ± 2 °C. Afterwards, the 4th instar larvae were visually detected by a relatively larger size, and larvicidal activity was performed following the WHO protocol¹⁹. Finally, % mortality and LD50 values were determined.

Gas chromatography-mass spectrometry (GCMS) analysis

For GC/MS, 70 µL of plant oil was accumulated by adding 10 grams of powdered plant material in 100 mL of ultrapure dH2O and then performing microwave-assisted hydro-distillation for 40 min. GCMS instrument (Agilent technologies - GC7890B and MS5977A) was equipped with DB-5MS fused capillary column. Various compounds were recognized with the help of NIST and WILEY database^20,21.

Statistical analysis

All assays were performed twice and mean ±SD were calculated. The least significant difference (LSD) was observed using Statistix 8.1, and a P-value (< 0.05) was used to establish statistical significance.

Plant extraction

The polarity of extraction solvents plays a critical role in plants’ biological activities²². In this study, DMSO and glycerol were chosen as green solvents in combination with the strongly polar solvent (methanol) to determine the phytochemicals and biological activities of five species.

Total phenolics, flavonoids and alkaloids content

Our study showed a large variation in TPC, TFC and TAC among selected extracts. TPC and TFC were observed highest in S. mollis (32.35 ± 0.19 mg gallic acid equivalent/g and 15.00 ± 0.78 mg rutin equivalent/g) and M. pruriens (31.62 ± 0.22 mg GAE/g and 16.66 ± 0.78 mg RE/g) methanol extracts while lowest in the methanol-glycerol extracts of P. tuberosa (7.75 ± 0.26 mg GAE/g and 3.33 ± 0.78 mg RE/g) and M. pruriens (8.05 ± 0.07 mg GAE/g and 3.05 ± 0.39 mg RE/g) (Figure 1).

Similarly, the highest TAC was also recorded in SMM (9.61 ± 0.54 mg AE/g) and MPM (9.61 ± 0.54 mg AE/g) and lowest in MPMG (2.69 ± 0.54 mg AE/g) and PTMG (3.07 ± 0.00 mg AE/g). However, all phytochemical contents were found in descending order of methanol extracts > methanol-DMSO extracts > methanol-glycerol extracts.

Figure 1 Phytochemicals observed in five Fabaceae extracts. Data represents mean ± SD and alphabetical values (a-k) are significantly different by P < 0.05. 

Antioxidant activity

The antioxidant activity revealed DPPH activity in descending order of methanol extracts > methanol-DMSO extracts > methanol-glycerol extracts. The lowest IC50 value was recorded in MPM, i.e. 14.09 ± 3.60 µg/mL, indicating the highest scavenging activity, while the highest IC50 value was found in PTMG, i.e. 1772.66 ± 10.01 µg/mL showing the lowest antioxidant capacity (Figure 2a). The correlation test showed that DPPH activity was strongly correlated with TPC (r = 0.845), TFC (r = 0.842) and TAC (r = 0.842) (Figure 2b). It can be suggested that the DPPH scavenging activity of leaf extracts is directly associated with the concentration of phenolic, flavonoid and alkaloid content in leaves. Similarly, our results are comparable to the other studies that TPC and DPPH activity were 10.96 ± 0.21 mg GAE/g and 61.51 ± 0.33 % in S. sesbans, 2.89 ± 0.01 mg GAE/g and 28.21 ± 1.29 % in L. purpureus and 39.96 ± 0.00 mg GAE/g and 40-60 % in M. pruriens grown in Thailand and India(23, 6).

Figure 2 Antioxidant activity of plant extracts assessed at different concentrations and its correlation with phytochemicals (a) IC50 values determined in DPPH assay (b) Correlation between DPPH activity and phytochemicals. 

DNA damage protection assay

In this assay, untreated plasmid expressed two bands while strand scission was observed on treating DNA with Fenton reagent. The gel photographs showed that PTM (3631.88) and SSMD (2050.85) exhibited highest protection, whereas SSM (1283.59), MPM (1088.60), SMM (1044.59), PTMD (886.41) and SSMG (211) possesses lowest protection against oxidative DNA damage, as corroborated by densitometric analysis (Figure 3b). However, all other extracts were found to be ineffective in protecting DNA from damage (Figure 3a). In the current study, methanol extracts protected DNA by scavenging the oxidation products that damage the DNA. It can be inferred that the abundance of TPC, TFC and TAC present in leaves acted directly on the oxidative agents and prevented the chain reaction of oxidative stress which alleviated oxidative damage in the methanol extracts.

Figure 3 Effects of Fabaceae extracts showing DNA damage protection ability (a) Gel photograph displaying DNA bands (Lane 1: plasmid; Lane 2: plasmid + reagent + standard; Lane 3: plasmid + reagent; Lane 4: plasmid + reagent + MPM; Lane 5: plasmid + reagent + SSM; Lane 6: plasmid + reagent + SMM; Lane 7: plasmid + reagent + PTM; Lane 8: plasmid + reagent + LPM; Lane 9: plasmid + reagent + MPMD; Lane 10: plasmid + reagent + SSMD; Lane 11: plasmid + reagent + SMMD; Lane 12: plasmid + reagent + PTMD; Lane 13: plasmid + reagent + LPMD; Lane 14: plasmid + reagent + MPMG; Lane 15: plasmid + reagent + SSMG; Lane 16: plasmid + reagent + SMMG; Lane 17: plasmid + reagent + PTMG; Lane 18: plasmid + reagent + LPMG) (b) Densitometry calculation of DNA bands. 

α-Amylase and α-glucosidase inhibition assays

In current study, α-amylase assay revealed lowest IC50 value in SMM (10.62 ± 7.71 µg/mL) and SSM (17.01 ± 11.94 µg/mL) indicating highest α-amylase inhibition potential (Figure 4a). In α-glucosidase assay, highest activity was recorded in the methanol-glycerol and methanol-DMSO extracts of S. sesbans (i.e. 0.02 ± 0.01 µg/mL and 0.03 ± 0.03 µg/mL IC50 values). In contrast, lowest α-glucosidase inhibitory potential was found in all extracts of P. tuberosa (IC50 values 13.82 to 26.36 µg/mL) (Figure 4c). Comparatively, α-amylase activity was significantly correlated with TPC (r = 0.490) and TFC (r = 0.470) as compared to the TAC (r = 0.394) (Figure 4b). Likewise, α-glucosidase inhibitory activity also depicted moderately positive correlation with TPC (r = 0.497), TFC (r = 0.446) and TAC (r = 0.483) (Figure 4d). The r value of 0.4 indicates a moderately positive correlation but not a stronger one. The results further corroborate previous studies²² that elucidated > 80 % α-glucosidase inhibitory activity in S. sesbans and L. purpureus extracts. Similarly, Gulati et al. ⁴ documented significantly lower IC50 values for both α-amylase (< 25 µg/mL) and α-glucosidase (< 5 µg/mL) inhibitory activities in M. pruriens grown in Ethiopia.

Figure 4 Antidiabetic activity of plant extracts and its correlation with phytochemicals (a) α- amylase inhibition assay (b) Correlation of α- amylase inhibition activity with phytochemicals (c) α- glucosidase inhibition assay (d) Correlation of α- glucosidase inhibition activity with the phytochemicals. * r is correlation coefficient and IC50 stands for half-maximal inhibitory concentration. * Acarbose was used as a standard. 

Anti-leishmanial and anti-dengue assays

All examined species showed significant biological activities in their methanol extracts. Hence, the methanol extracts were further tested to examine anti-leishmanial and anti-dengue potential of these species. The anti-leishmanial activity was found in descending order of S. sesbans (82.99 % inhibition) > L. purpureus (69.31 % inhibition) > M. pruriens (68.85 % inhibition) > P. tuberosa (66.92 % inhibition) > S. mollis (36.27 % inhibition) (Table 2). In the anti-dengue assay, only S. sesbans and L. purpureus were found to be effective in controlling the growth of A. aegypti as they displayed LD50 values of 70.10 and 200.00 ppm, respectively (Table 3).

Previously²⁴, determined anti-plasmodial flavones from the roots of S. mollis. Hence, the antileishmanial activity of leaves examined in the current study was ineffective in inhibiting leishmanial parasites’ growth. The current study confirmed the parasite inhibitory potential of M. pruriens using L. major promastigotes.

The decreased parasite growth can be ascribed to the increased production of ROS and reduced level of arginase that can be metabolized to nitric oxide (NO), a microbicidal agent responsible for the intracellular parasite removal²⁵.

GC/MS analysis

A total of 37 compounds, viz. hydrocarbons, fatty acids, alcohols, esters and carbohydrates, were identified, and differences in their peak area were observed in the GC/MS chromatograms. Among these, 9-octadecenamide, (Z) and tetradecanamide were found to be dominant compounds ranging from 11.94 to 63.89 % in all species. Similarly, 13-Docosenamide was also present in higher concentration (20.87 %) in L. purpureus only. However, all other compounds were found < 12 % as shown in Table 4.

The 9-octadecenamide (Z) is used as a hypolipidemic agent and for treating atherosclerosis, while tetradecanamide is considered for anti-mycobacterial and anti-tubercular activities²⁶. Previously²⁷, proposed anti-leishmanial properties of 13-docosenamide, which have been detected significantly (20.87 %) in L. purpureus leaves. Cis-11-eicosenamide (12.03 %) was detected only in P. tuberosa and hexadecanoic acid-methyl ester was recorded in minor concentration in S. sesbans (0.55 %) only.