Polarity based characterization of biologically active extracts of Ajuga bracteosa Wall. ex Benth. and RP-HPLC analysis

Abstract

Background: The concept of botanical therapeutics has revitalized due to wide importance of plant derived pharmaceuticals. Therefore, the ameliorative characteristics of Ajuga bracteosa were studied.

Methods: Total phenolic content, flavonoid content, antioxidant capacity, reducing power and free-radical scavenging activity were determined colorimetrically. Specific polyphenols were quantified by RP-HPLC analysis. Preliminary cytotoxicity was tested using brine shrimp lethality assay while antiproliferative activity against THP-1 and Hep-G2 cell lines was determined by MTT and SRB protocols respectively. Antileishmanial potential was assessed via MTT colorimetric method. To investigate antidiabetic prospect, α-amylase inhibition assay was adopted whereas disc diffusion method was used to detect likely protein kinase inhibitory, antibacterial and antifungal activities.

Results: Among fifteen different extracts, maximum total phenolic content (10.75 ± 0.70 μg GAE/mg DW), total reducing power (23.90 ± 0.70 μg AAE/mg DW) and total antioxidant capacity (11.30 ± 0.80 μg AAE/mg DW) were exhibited by methanol extract with superlative percent extract recovery (17.50 ± 0.80% w/w). Chloroform-methanol extract demonstrated maximum flavonoid content (4.10 ± 0.40 μg QE/mg DW) and ethanol extract exhibited greatest radical scavenging activity (IC₅₀ 14.40 ± 0.20 μg/ml). RP-HPLC based quantification confirmed polyphenols such as pyrocatechol, gallic acid, resorcinol, catechin, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, vanillic acid, coumarin, sinapinic acid, trans-cinnamic acid, rutin, quercetin and kaempferol. The brine shrimp lethality assay ranked 78.60% extracts as cytotoxic (LC₅₀ ≤ 250 μg/ml) whereas significant THP-1 inhibition was shown by methanol-acetone extract (IC₅₀ 4.70 ± 0.43 μg/ml). The antiproliferative activity against Hep-G2 hepatoma cancer cell line was demonstrated by n-hexane, ethylacetate and methanol-distilled water (IC₅₀ 8.65-8.95 μg/ml) extracts. Methanol extract displayed prominent protein kinase inhibitory activity (MIC 12.5 μg/disc) while n-hexane extract revealed remarkable antileishmanial activity (IC₅₀ 4.69 ± 0.01 μg/ml). The antidiabetic potential was confirmed by n-hexane extract (44.70 ± 0.30% α-amylase inhibition at 200 μg/ml concentration) while a moderate antibacterial and antifungal activities were unveiled.

Conclusion: The variation in biological spectrum resulted due to use of multiple solvent systems for extraction. We also deduce that the valuable information gathered can be utilized for discovery of anticancer, antileishmanial, antioxidant and antidiabetic bioactive lead candidates.

Keywords: Ajuga Bracteosa; Antiproliferative activity; Hep G2 hepatoma cell line; Leishmania tropica; Protein kinase inhibition; THP-1 human leukemia cell line.

Fig. 1

Percent extract recovery of A. bracteosa using mono and binary (1:1) solvents for extraction. Nh: n-hexane, Nh-Ea: n-hexane-ethyl acetate, Nh-E: n-hexane-ethanol, C: chloroform, Ea: ethyl acetate, C-M: chloroform-methanol, Ea-M: ethyl acetate-methanol, A: acetone, M: methanol, A-M: acetone- methanol, A-E: acetone-ethanol, E: ethanol, A-Dw: acetone-distilled water, M-Dw: methanol-distilled water, Dw: distilled water

Fig. 2

Total phenolic content (μg GAE/mg DW), total flavonoid content (μg QE/mg DW), total reducing power (μg AAE/mg DW), total antioxidant capacity (μg AAE/mg DW) and free radical scavenging activity (%) of A. bracteosa in different extracts. IC₅₀ of ascorbic acid is found to be 21.8 μg/ml. Values are presented as mean ± Standard deviation (n = 3). The columns with different superscript (^a-g) letters show significantly (P < 0.05) different means

Fig. 3

RP-HPLC chromatograms of standard compounds (a) and ethyl acetate (b), water (c), and methanol (d) extracts of Ajuga bracteosa. Individual peaks showing phenolic compounds i.e. Hydroquinone (1), Pyrocatechol (2), Gallic acid (3), Resorcinol (4), Catechin (5), Chlorogenic acid (6), Caffeic acid (7), Vanillic acid (8), p-Coumaric acid (9), Ferulic acid (10), Sinapic acid (11), Coumarin (12), Salicylic acid (13), Trans cinnamic acid (14), Rutin (15), Quercetin (16), Ellagic acid (17) and Kaempferol (18)

Fig. 4

Antileishmanial activity of A. bracteosa in different solvents. Extracts and the standard are tested at the concentration of 100 and 0.33 μg/ml respectively. Values are presented as mean ± Standard deviation (n = 3). The columns with different superscript (^a-g) letters show significantly (P < 0.05) different means

Fig. 5

α-amylase inhibition assay of A. bracteosa extracts. Acarbose is used as a standard with an IC₅₀ 33.73 μg/ml. Values are presented as mean ± Standard deviation (n = 3). The columns with different superscript (^a-d) letters show significantly (P < 0.05) different means