Limonoids from selected local citrus fruits : quantification, isolation, characterization and evaluation of Phase II detoxifying enzyme-inducing and anti-inflammatory activities

Date

6-2012

Degree

Bachelor of Science in Chemistry

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Evelyn B. Rodriguez

Restrictions

Restricted: Not available to the general public. Access is available only after consultation with author/thesis adviser and only to those bound by the confidentiality agreement.

Abstract

The major limonoids from some selected local citrus fruits were quantified, isolated, characterized, and evaluated for their phase II enzyme-inducing and anti-inflammatory activities. Extraction of limonoids from pomelo seeds afforded two limonoid isolates: isolate 1 (5.5:4.5 limonoid A/ limonoid B) and isolate 2 (2:8 limonoid A/ limonoid B), with 0.52% and 0.37% yield, respectively. Fractionation by silica gel 60 open column chromatography (7:1 dicholoromethane/ethyl acetate) led to the isolation of pure limonoids A and B, identified as limonin and nomilin, respectively. The identities were confirmed by melting point determination, derivatization with p-dimethylaminobenzaldehyde, and UV-Vis and IR spectral analyses. The limonoid content of some selected local citrus fruits were determined by thin layer chromatography coupled with densitometry using limonoid isolate 1 as reference. Pomelo seed extract showed the highest limonin (336.0 ± 18.3 mg/ 100 g sample) and nomilin (209.1 ± 1.1 mg/ 100 g sample) content. Among the fruit extracts, pomelo fruit has the highest limonoid content with 244.6 ± 12.9 mg limonin/100 g sample and 175.4 ± 5.7 mg nomilin/100 g sample, followed by dayap fruit with 53.1 ± 2.9 mg limonin/100 g sample and 137.4 ± 8.3 mg nomilin/100 g sample. Lastly, dalanghita fruit showed the lowest level of limonin (4.0 ± 0.3 mg /100 g sample) and nomilin (16.6 ± 2.0 mg /100 g sample). The phase II enzyme-inducing activity of the limonoid isolates and pomelo seed extract were evaluated spectrophotometrically by observing the increase in the activity of glutathione S-transferases (GST) isolated from onion on the conjugation of reduced glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB). GSTs are major phase II detoxifying enzymes that detoxify and neutralize electrophilic molecules by mediating their conjugation with GSH. Results showed a dose-dependent GST-inducing activity for the limonoid isolates and the pomelo seed extract. The highest induction of GST against CDNB at P≤ 0.05 was observed for limonoid isolate 2 at 75 ppm (2.88 ± 0.22) followed by the 25 ppm dosage (2.01 ± 0.18). The 75 ppm dosage of limonoid isolate 1 showed induction ratio of 1.95 ± 0.32 while the pomelo seed extract showed lower inducing activities with respect to both the two limonoid isolates in all concentrations. The anti-inflammatory potential of the limonoid isolates and extracts at different dosages (15, 30, 75, 150 µg) were also determined using the duck embryo chorioallantoic membrane irritation assay. Hydrocortisone at 150 µg dosage showed the highest percent inflammation inhibition (99.56%) followed by the 150 µg dosages of limonoid isolate 2 (94.36 ± 0.76%) and limonoid isolate 1 (89.36 ± 1.06 %). Limonoid isolates 1 and 2 showed medium (at 15 and 30 µg dosage) to high (75 and 150 µg dosage) inflammation inhibition while the extract showed low to medium anti-inflammatory potential. The study demonstrated that limonoids isolated from pomelo seeds have significant GST-inducing and anti-inflammatory activities, indicating that the isolated limonoids may be useful in chemoprevention and in reducing the risk of inflammation-related diseases.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 2012 C42 /S25

Document Type

Thesis

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