Collection of plant and procurement of chemicals
The leaves of Luffa cylindrica Linn. (Cucurbitaceae) were collected in December 2015 from Ambajogai, Maharashtra, India. The plant was authenticated from Postgraduate Botany Department, N.E.S. Science College, Nanded, as Luffa cylindrica (L.) (Cucurbitaceae) with a voucher specimen no: S-1/21/02/15. Required chemicals were brought from a local market manufactured at the Qualigens Fine Chemicals, Mumbai.
Experimental animals
Albino Wistar rats of either sex or weight between 150 and 200 g were used from the animal house of the Nanded College of Pharmacy, Nanded, Maharashtra. Animals were housed in a group of four in separate cages and maintained at controlled conditions of temperature (28 ± 2 °C). The standard pellet food and purified drinking water were provided with free access. After completion of the experimental procedure, all the animals were sacrificed with euthanasia using ether up to their normal death using inhalation anesthetic.
Preparation of extract
The leaves of Luffa cylindrica (L.) were thoroughly washed with running tap water 2–3 times. After the shade drying, coarsely powdered, the material was used for extraction. Five hundred grams of the material was extracted using solvents with different polarity, namely methanol, ethanol, and chloroform by soxhlet extraction. Extraction was carried out in 48 cycles, and every cycle was of 1 h each so the total extraction was carried for 48 h. The extract was filtered and dried on a water bath and stored at room temperature. The methanolic extract was chosen for the animal study due to the presence of flavonoids, polyphenols, tannin, and saponins [13].
Phytochemical screening
Phytochemical screening was carried out to determine the presence of different phytochemicals. The standard procedures were performed for the determination of different phytochemicals. After performing the test with specific reagents, the inference was given by visual observation of color change or precipitate formation [14].
Acute toxicity studies
Acute toxicity of methanolic extract of Luffa cylindrica (L.) leaves was determined using male Swiss albino rats as per OECD guideline 423. After overnight fasting, the weight of the individual rat was recorded before the study. Animals were divided randomly into four treatment groups; each group consisting of three rats and each treatment group receives orally the methanolic extract of Luffa cylindrica (L.) leaves in a dose of 2000 mg/kg. Close observation of animals was done for 4 h after the administration of the extract, and further observation was done up to 14 days to notice if any change occurs in general behavior and/or other physical activities [15].
Antihyperlipidemicactivitystudy of Luffa cylindrica (L.) leaf extract on Triton X-100-induced rats
The Wistar rats were randomly divided into 5 groups, and each group was comprised of 6 rats. A standard pellet diet was given to the first group along with water and 5% CMC was administered orally. The animals from the II, III, IV, and V groups were intraperitoneally injected with 10% aqueous solution of Triton X-100 (400 mg/kg body weight). Five percent CMC (p.o) was given to the second group for 7 days after 72 h of administration of Triton X-100 injection. MELCL was daily administered to the third and fourth group with the doses of 100 and 200 mg/kg using 5% CMC, as a vehicle, per oral (p.o) for 7 days, after inducing hyperlipidemia. Atorvastatin was administered to the fifth group with a dose of 10 mg/kg, p.o for 7 days. Before blood sampling for biochemical estimation, the food was withdrawn 10 h prior, and on the 8th-day, biochemical estimation was performed [16].
Biochemical estimation
The blood samples were collected on the 8th day for all the groups by the retro-orbital puncture after sacrificing the animal with inhalation ether euthanasia. After the clotting of blood, the centrifugation was carried out at 3000 rpm for 10 min. The serum sample was subjected to biochemical analysis for the determination of serum levels of triglycerides, total cholesterol, low-density lipoprotein cholesterol, very-low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol. The determination of biochemical parameters was carried out as per the standard procedure provided by the manufacturer in the instruction manual of the auto analyzer kit (Hitachi 704 analyzer) [17].
Antioxidant potential of Luffa cylindrica (L.) leaf extract
DPPH (2,2-diphenyl, 1,1-picrylhydrazyl) radical scavenging activity
The solution of DPPH was prepared by dissolving 1.97 mg of DPPH in 50 ml methanol. The sample solution of extract was prepared by dilution method by diluting the extract up to 100 μg/ml. 0.2 ml of the sample solution of MELCL was taken and the standard (ascorbic acid), at a concentration of 50, 100, 150, and 200 μg/ml was added. One milligram of 0.1 mM freshly prepared DPPH radical solution was added. After vigorous shaking, the reaction mixture is allowed to stand for 30 min at room temperature. The control mixture contains all the reagents except the extract and is considered as a blank. An ultraviolet-visible spectrophotometer (UV-1800, SHIMADZU, Japan) was used to measure the absorbance of all reaction mixtures at 517 nm. The DPPH radical scavenging potential (%) of the sample was calculated with the following formula:
$$ \%\mathrm{Inhibition}={\mathrm{A}}_{\mathrm{blank}}-{\mathrm{A}}_{\mathrm{sample}}/{\mathrm{A}}_{\mathrm{blank}}\times 100 $$
(1)
where Ablank is the absorbance of ascorbic acid and A sample is the absorbance of extract. All tests were performed in duplicate [18].
Hydrogen peroxide radical scavenging activity
Hydrogen peroxide solution (40 mmol/L) was prepared using phosphate buffer (pH 7.4) as a vehicle. The concentration of hydrogen peroxide was determined at 230 nm using a UV-visible spectrophotometer (UV-1800, SHIMADZU, Japan). Test samples (50, 100, 150, 200, and 250 μg) were added to a hydrogen peroxide solution (0.6 ml, 40 mmol/L). Absorbance was detected at 230 nm and was determined after 10 min against a blank solution. The scavenging of hydrogen peroxide by the sample and ascorbic acid was calculated using the same equation as above [19].
Statistical analysis
The statistical analysis of data was carried out using InStat-GraphPad (3.0). The values are expressed as mean ± SEM (n = 6) along with ANOVA followed by Dunnett test.