Extracts preparation

Fresh leaves of A. julibrissin subsp. Julibrissin and C. decapetala (Roth) Alston were collected and their accession numbers were obtained from the Herbarium of National Agriculture Research Center (NARC), Islamabad. The weight of extracts was measured to ascertain extract yield as shown in Table 1.

Acetylcholinesterase inhibitory assay

The acetylcholinesterase inhibition potential was assessed using 140 µL of 0.1 M sodium phosphate buffer (pH 8) was added in 20 µL of plant extract, 15 µL of AChE enzyme solution (0.2 units/ml) and 10 µL of 15 mM DTNB followed by the incubation for 10 min at room temperature. The absorbance of the final product was measured at 412 nm and galantamine (100, 500 and 1000 µg/ml) was measured as a positive control. The enzyme inhibition was determined as

Where V_max is an enzyme activity without inhibitor.

Lipase inhibitory assay

Briefly, 5 mg/ml lipase from porcine pancreatic type II (Sigma) was added to 50 mM Tris-HCl buffer (pH 8.0) and then centrifuged for 5 min at 5500 rpm. Then, 100 µL of lipase solution was added to 50 µL of plant extract (100, 500 and 1000 µg/ml prepared in DMSO) and left for incubation on ice and the absorbance was noted at 405 nm. Orlistat was measured as a standard and the inhibition potential was calculated ¹⁵.

Anti-glycation assay

The AGE inhibition in plant extracts was determined using the method¹⁶. The fluorescence was measured at the emission wavelength of 440 nm and the percentage inhibition of AGE formation was calculated as:

Cytotoxicity assay against various cell lines

Anti-acetylcholinesterase assay

Plants are the potential source of compounds that can prevent or treat neurodegenerative diseases by inhibiting acetylcholinesterase²⁰. The present study revealed highest acetylcholinesterase inhibitory activity in A. julibrissin methanolic extract (IC₅₀ 2.391 µg/ml) followed by A. julibrissin methanol-DMSO extract (IC₅₀ 10.16 µg/ml) and C. decapetala methanolic extract (IC₅₀ 22.79 µg/ml). However, C. decapetala methanol-DMSO extract showed lowest anti-acetylcholinesterase activity by displaying IC₅₀ value of 30.24 µg/ml (Table 2).

The AChE inhibition potential of these extracts could be attributed to the existing phenolic and flavonoid compounds ⁽²¹. A previous report ²² indicated anti-acetylcholinesterase activity in the ethanolic leaves extracts of Albizia lucidor (IC₅₀ 24.89 ± 1.60 µg/ml) and Albizia procera (IC₅₀ 43.50 ± 2.10 µg/ml). In another study, ²³ demonstrated highest inhibition potential (10.20 mg galantamine equivalent/g) of C. decapetala leaves extracts against butyrylcholinesterase. However, the acetylcholinesterase inhibitory activity of selected species has been examined for the first time. It can be inferred that the variations within the same plant species could be due to the different phyto-constituents of the plants that are grown in various geographical areas in different seasons of the year. The phyto-constituents may vary depending on the soil, water, stage of plant and the time of collection ⁽²⁴. It can be suggested that A. julibrissin and C. decapetala leaves may serve as an ideal candidate for designing new AChE inhibitors to treat neurodegenerative disorders. However, detailed in vivo tests are needed to validate the efficiency of these species for medicinal use.

Anti-lipase and anti-glycation assays

The inhibition of pancreatic lipase (a lipolytic enzyme) and glycation products is a highly effective method that protects against fat absorption in individuals with obesity and hyperglycemia²⁵. In this study, pancreatic anti-lipase and anti-glycation potential of two species have been investigated and results are presented in Figure 1a and b. The highest lipase and glycation inhibitory potential was observed in C. decapetala methanolic extract (IC₅₀ 114.9 µg/ml and 43.69 µg/ml) followed by the methanol-DMSO extract of A. julibrissin (IC₅₀ 138.7 µg/ml and 74.06 µg/ml). However, weak anti-lipase and anti-glycation activity was recorded in A. julibrissin methanolic extract (IC₅₀ 155.2 µg/ml and 182.5 µg/ml) and C. decapetala methanol-DMSO extract (IC₅₀ 168.4 µg/ml and 247.3 µg/ml) respectively (Table 2). The remarkable inhibitory activity of selected extracts indicates their potential use as a powerful source of anti-obesity and anti-glycation agents.

Previously, researchers have investigated biological potential of different species of Albizia and Ceasalpinia grown in different regions of world. For instance, ⁽²⁶ demonstrated pancreatic lipase inhibition potential in Caesalpinia sappan commonly grown in Thailand. Contrarily, ⁽²⁷⁾ observed no lipase inhibitory potential in Albizia lebbeck that was grown in Thailand. It can be inferred that species belonging to the same family, irrespective to different climatic conditions, exhibit potent anti-lipase and anti-glycation activities. The relative bio-efficacy can be ascribed to the presence of phenolic and flavonoid compounds as suggested in previous literature ^(28-29. However, the plant extract is a mixture of bioactive compounds that contribute to the bioactivities altogether. This suggests that the composition of active compounds, structural features and other potential factors in plant extract may have also affected pancreatic lipase and hyperglycemic conditions. Therefore, the exact relationship between the active compounds and biological activities needs to be further investigated.

Meth. stands for methanol and DMSO stands for dimethyl sulfoxide; Values in tables are presented as mean ± SD (n=3); IC₅₀ value indicates half maximal inhibitory concentration.

Orlistat was used as a control in anti-lipase assay and rutin was used as a control in anti-glycation assay.

Figure 1: Anti-lipase and anti-glycation activity of leaves extracts of selected species (a) Anti-lipase assay (b) Anti-glycation assay. *Data indicates mean ± SD (3n) and each alphabetical letter (a-g) indicates a significance difference at P < 0.05. *Orlistat was used as a control in anti-lipase assay and rutin was used as a control in anti-glycation assay. 

Cytotoxic activity

As the methanolic extracts displayed significant biological activities, hence were further tested to observe there in vitro cytotoxic potential against three different cell lines. Among different plant extracts, C. decapetala showed highest cytotoxic potential against PC3 and 3T3 cell lines by showing IC₅₀ value of 144.3 ppm and 201.0 ppm. Contrarily, the lowest activity was recorded in A. julibrissin methanolic extract against PC3 cell lines (IC₅₀ 459.8 ppm) and 3T3 cell lines (IC₅₀ 392.6 ppm). However, the moderate inhibitory potential was observed in both species when tested against HeLa cell lines. Overall, C. decapetala leaf extract displayed the highest cytotoxic potential compared to A. julibrissin (Table 3).

Previously, ⁽³⁰⁾ isolated emodin, baicalein and apigenin from C. decapetala roots and confirmed their anti-tumor activities against human gastric carcinoma cell line MGC-803 cell line with IC₅₀ values of 15.6, 16.3 and 13.2 µmol/L using MTT assay. Our studies also corroborate the earlier findings ²³ who reported highest cytotoxicity (i.e. 46.08 µg/ml CC₅₀) for the bark methanol extract of C. decapetala on the HeLa cells. Moreover, previous researcher ³¹⁾ isolated oleanane-type saponins, julibrosides, from the stem bark of A. julibrissin and examined their cytotoxic effects against HCT-116, BGC-823, HepG2 and A549 cell lines. Earlier studies ^32,33 revealed anti-cancer activity of another specie of Albizia genus i.e. Albizia lebbeck using MCF-7 (human breast cancer), HeLa and A549 cell lines. However, A. julibrissin leaves extracts have been examined for the first time against selected cell lines. The anti-carcinogenic potential of examined species could be attributed to the active compounds such as Julibroside saponins present in A. julibrissin³⁴. In general, leaves of selected species showed promising results as a cytotoxic agent due to the relatively high toxicity on selected cells. Hence, it can be inferred that the exact mechanism of apoptosis should be investigated using flow cytometry and microscopy techniques.

HPLC análisis

In current study, HPLC method was used to quantify six compounds present in the leaves of selected species. The obtained HPLC chromatograms are presented in Figure 2 (a to d) which indicated that A. julibrissin possesses highest concentration of chlorogenic acid (56.06 ppm) and quercetin dihydrate (15.71 ppm) while C. decapetala leaves extracts exhibit the highest concentration of gallic acid (73.55 ppm). However, all other compounds were detected in lower concentrations (i.e. below 12 ppm) in these species (Table 4).

Figure 2: HPLC chromatograms indicating six phenolic and flavonoid compounds at respective retention times in selected species (a) Gallic acid, vanillic acid, chlorogenic acid, rutin trihydrate and quercetin dihydrate in A. julibrissin (b) Caffeic acid in A. julibrissin (c) Gallic acid, vanillic acid, chlorogenic acid, rutin trihydrate and quercetin dihydrate in C. decapetala (d) Caffeic acid in C. decapetala. 

Previously, scientists ¹³ showed the presence of gallic acid, quercetin, catechin, 4-hydroxybenoic acid and p-coumaric acid as the main phenolic compounds in C. decapetala extracts using the HPLC technique which has been confirmed in the current study. Similarly, ³⁵ reported eight compounds in C. decapetala using UPLC-MS/MS. Furthermore, ^(36-37 indicated the presence of lignans, triterpenoids saponins and some phenolic compounds in the stem bark of A. julibrissin. Besides, cassane diterpenoid, caesaldecan, squalene, lupeol, resveratrol, quercetin, stigmasterol and astragalin, the main phytochemicals observed in C. decapetala are terpenoids, flavonoids and tannins³⁸. It can be inferred that the selected species possesses a valuable reservoir of polyphenolic compounds of pharmacological significance and thus needs to be isolated and investigated for food and industrial application.