Plant material
A. lacucha was collected from Sonargoan, Narrayangonj, Bangladesh in October 2015. The plant materials including bark were authenticated by Mr. Ahsan Habib, Senior Scientific Officer, Bangladesh National Herbarium, Mirpur, Dhaka. A voucher specimen of the plant material was deposited in the herbarium under the number, DACB: 42083, for future reference. The bark of the plant was shade dried and grounded to a coarse powder. One kilogram of the bark powder was mixed with 4000 mL methanol-water (80:20) for 10 days. The extract was filtered using cotton bed followed by filter paper (Whatman No. 1) and the filtrate was concentrated using a rotary evaporator at 40 °C and 50 r.p.m. The concentrated filtrate was subjected to lyophilization after freezing. Finally, 82 g of dried extract (yield 8.20%) was obtained.
Preliminary phytochemical screening
The plant extract was subjected to qualitative phytochemical screening for the detection of carbohydrates, glycosides, saponins, flavonoids, tannins, alkaloids, resins and steroids, by the methods described by Ghani (2003) [9]. Total flavonoid and phenolic contents of the extract were determined by using aluminum chloride (AlCl3) and Folin–Ciocalteu’s reagent according to the procedure described by Selim et al. (2014) [20] and Singleton et al. (1999) [21] respectively.
Isolation and identification of compound
Thirty-five grams of bark extract was subjected to fractionation by Kieselgel (60–120 mesh, 710 g) vacuum liquid chromatography (VLC) using n-hexane, ethyl acetate (EtOAc) and methanol (MeOH). The collected fractions (41 fractions, 150 mL each) were analyzed by thin layer chromatography (TLC) and the similar fractions were combined together into five fractions (Fraction 1–5). After analysis the of combined fractions by TLC, fraction 2 (93% EtOAc in MeOH, 14.74 mg) was subjected to preparative thin layer chromatography over 60 F254, 20 × 20 cm PTLC plate which was developed with CHCl3: MeOH (4:1). The developed bands on the TLC plate was observed under UV light (254 nm and 356 nm) and was sprayed with 1% vanillin-sulfuric acid followed by heating at 110 °C. The selective band, with a Rf value of 0.15, was separated from the plate, eluted with ethyl acetate-chloroform (1:1), and chloroform-methanol (1:1). The solvents were dried to yield a pure compound (8 mg). The structure of the compound (Fig. 1) was elucidated as (+)-catechin by comprehensive analysis of 1H, 13C, DEPT, COSY, HSQC and HMBC NMR spectra (Additional file 1) and was confirmed by comparing the respective published spectral data [22, 23].
(+)-Catechin: pale yellow powder, m.p. 174–76 °C. 1H NMR (400 MHz, CD3OD): δH = 4.56 (1H, d; J = 7.60 Hz; H-2), 3.97 (1H, ddd; J = 5.20, 8.00, 7.60 Hz; H-3), 2.86 (1H, dd; J = 5.20, 16.00 Hz; H-4α), 2.52 (1H, dd; J = 8.00, 16.00 Hz; H-4β), 5.92 (1H, d; J = 1.80 Hz; H-6), 5.85 (1H, d; J = 1.80 Hz; H-8), 6.83 (1H, d; J = 1.20 Hz; H-2′), 6.76 (1H, d; J = 8.00 Hz; H-5′) and 6.72 (1H, dd; J = 8.00, 1.20 Hz; H-6′). 13C NMR (100 MHz, CD3OD): δC = 81.5 (C-2), 67.4 (C-3), 27.1 (C-4), 156.2 (C-5), 94.9 (C-6), 156.5 (C-7), 94.2 (C-8), 155.5 (C-9), 99.5 (C-10), 130.9 (C-1′), 113.9 (C-2′), 144.9 (C-3′), 144.9 (C-4′), 114.7 (C-5′) and 118.7 (C-6′).
Chemicals and standard drugs
Methanol, chloroform, n-hexane, ethyl acetate, toluene, vanillin, sulfuric acid, acetic acid, and formalin were purchased from Merck Co. (Darmstadt, Germany). Methanol-d4 (CD3OD), sodium carboxymethylcellulose (Na-CMC), λ-carrageenan, (+)-catechin and pentobarbital sodium were purchased from Sigma-Aldrich (St. Louis, MO, USA). The standard drugs including, morphine sulfate (Gonoshasthaya Pharmaceuticals Ltd., Bangladesh), diclofenac sodium (Novartis Bangladesh Ltd., Bangladesh), naloxone hydrochloride (Samarth Life Sciences Pvt. Ltd., India), glibenclamide, diazepam (Square Pharmaceuticals Ltd., Bangladesh) were either purchased or was obtained as gifts.
Animals
The experimental animals, Swiss albino male mice (4–6 weeks old, 20–25 g), were procured from Animal Resources Branch of the International Center for Diarrheal Disease Research, Bangladesh (icddr,b). The animals were housed in wooden cages (120 × 30 × 30 cm) containing flake wood shavings as bedding. All the animals were kept under standard laboratory environmental condition with 12 h light/dark cycle (lights on at 6.00 a.m. and off at 6.00 p.m.), 25 ± 2 °C room temperature and 55–60% relative humidity. They were fed with standard pellet diet manufactured by icddr,b, and access was given to tap water ad libitum. For acclimatization, the animals were left for 14 days in the laboratory. Health status of every mouse was checked regularly. Before experiments, animals were randomly allocated into control, positive control and experimental groups (five animals per group, n = 5). Before 3–4 h of tests, access to food was restricted for the experimental animals but free access to water was ensured. Appropriate steps were taken to diminish their sufferings.
Treatments
The animals of control group received 0.5% Na-CMC by oral administration at the dose of 10 mL/Kg b.w., 30 min before experiments. For the positive control group mice, standard drug, morphine (5 mg/Kg b.w.) or diclofenac sodium (10 mg/Kg b.w.) was intraperitoneally (i.p.) administered 15 min before experiments. The experimental group mice received oral dose (50, 100 or 200 mg/Kg b.w) of catechin or hydro-alcoholic extract of the bark of A. lacucha (HEBA). The doses of (+)-catechin and HEBA were selected on the basis of trial experiments and their previously reported effective doses [18, 24, 25]. Naloxone (2 mg/Kg b.w., i.p.) and glibenclamide (10 mg/Kg b.w., i.p.) was used 15 min before morphine, HEBA or (+)-catechin administration. Na-CMC (0.5%, w/v, prepared in physiological saline) was used as a vehicle to prepare all the doses of standard drugs, experimental compound or extracts.
Acute toxicity test
The acute oral toxicity test of HEBA was carried out on six groups of mice (n = 5) at the dose of 50, 100, 500, 1000, 2000 and 3000 mg/Kg according to the principle of 420 – Fixed Dose Procedure of Organization for Economic Cooperation and Development (OECD). Control group mice (n = 5) received vehicle at the dose of 10 ml/Kg b.w, (p.o.). Both the experimental and control groups were provided with free access to food and water ad libitum. The mice were observed for mortality, allergic reactions, and any abnormal behaviors 14 days after treatment. On the 15th day the mice were sacrificed and their vital organs including heart, liver, lung, stomach, and kidneys were subjected to macroscopic evaluation [26].
Antinociceptive experiments
Hot plate test
The central nociceptive activity of HEBA and (+)-catechin was assessed using Eddy’s hot plate apparatus (Kshitij Innovations, Haryana, India), according to the method described by Eddy and Leimbach (1953) [27]. Animals were placed individually on the surface of the heated metal plate which was maintained at 55 ± 0.5 °C. Responses of the mice including forepaw licking, jumping, and withdrawal of paw(s) were considered as nociception. A pre-treatment latency of each mouse on the hot plate was recorded as a baseline. The latency times of experimental mice were recorded at 30, 45, 60, 90- and 120-min following treatments with vehicle, morphine, HEBA or (+)-catechin. Mice were kept on the hot plate for a maximum time period of 20 s to avoid any tissue injuries. The percentage of maximal possible analgesic effect (MPE) was calculated using the following formula: % MPE = [(post − treatment latency – pre − treatment latency)/(cut − off time – pre − treatment latency)] × 100.
Tail immersion test
Tail immersion test was performed according to the method previously described by D’Amour and Smith (1941) [28]. This experiment involved immersing 1–2 cm of the tail of each mouse into hot water bath maintained at 52 ± 1 °C. The mice were immobilized using ‘Chux’ for a few seconds. The latency time of tail withdrawal from hot water was measured and was used as an index of nociception. The tail of the mice was immersed for a maximum time period of 20 s to avoid tissue injuries. A latency was recorded before administration of vehicle, morphine, HEBA or (+)-catechin. The nociceptive reactions were recorded at 30, 45, 60, 90- and 120-min following treatments. The % MPE was measured using the formula as mentioned in the hot plate test.
Acetic acid-induced writhing test
The test was conducted according to the procedure described by Rauf et al. (2016) [29] with slight modification. Briefly, the writhing was induced by administrating 1% (w/v) acetic acid (10 ml/Kg, b.w., i.p.) after 30 min of vehicle, HEBA, (+)-catechin or 15 min of diclofenac sodium (10 mg/Kg, i.p.) administration. The individual mice were observed for nociceptive responses including contraction of the abdominal muscle and stretching of the hind limbs. The number of writhings were counted during a 30 min time period following the first response. The percentage inhibition of writhing was calculated using the following formula: % Inhibition = [{Mean no. of writhes (Control) − Mean no. of writhes (Test)}/{Mean no. of writhes (Control)}] × 100.
Formalin-induced nociception test
A formalin solution (5% formalin in 0.9% saline, 25 μL) was injected subcutaneously into the plantar surface of the right hind paw of each mouse to induce pain after 15 min of morphine and 30 min of vehicle, HEBA, (+)-catechin treatments. The licking, biting responses of the injected paw were considered as nociception. The time of responses were measured every 5 min, for a period of 45 min. Edema (Δ) of the injected paw was calculated from its thickness which was measured before and 60 min after the formalin administration using a digital slide caliper. The inhibition of the degree of edema was estimated according to following equation: Δ = (paw thickness after formalin injection – paw thickness before formalin injection) [30].
Carrageenan-induced paw edema test
The test was performed according to the procedure described by Winter et al. (1962) [31]. One hundred microliters of 1% (w/v) suspension of λ-carrageenan was administered into the sub-plantar region of the right hind paw of each mouse after diclofenac sodium, vehicle, HEBA or (+)-catechin administration. The paw thickness (mm) of each mouse was measured using digital slide caliper before (Cb) and at 0, 1, 2, 3, 4, 5, 6 h., after (Ca) carrageenan administration to determine the degree of edema (Δ). The percent inhibition of edema in was determined by using following formula: % Inhibition = [{(Ca − Cb)control – (Ca − Cb)test}/(Ca − Cb)control}] × 100.
Analysis of possible mechanism of action
Involvement of opioid receptors
The possible participation of opioid receptors in the antinociceptive effect of HEBA and (+)-catechin was investigated according the procedure described by Khan et al., (2011) [32]. Briefly, naloxone (2 mg/Kg, b.w., i.p.), a non-selective opioid receptor antagonist, was administered, 15 min before the treatments of HEBA or (+)-catechin in tail immersion and hot plate test. The latency times were recorded as discussed in the previous sections.
Involvement of ATP-sensitive K+ channel system
The possible involvement of ATP-sensitive K+ channel system in the antinociceptive effect of HEBA and (+)-catechin was determined according to the procedure described by Perimal et al. (2011) [33]. Mice received glibenclamide (10 mg/Kg, i.p.), an ATP-sensitive K+ channel blocker, 15 min before the treatments of HEBA or (+)-catechin. After 30 min of treatments, they were treated with 1% (w/v) acetic acid. The writhing response was counted as discussed in the acetic acid-induced writhing test.
Statistical analysis
Results are presented as mean ± SEM. Statistical analysis was performed by one-way or two-way analysis of variance (ANOVA) followed by Dunnett’s or Bonferroni’s test as appropriate post hoc test, using SPSS 22 software (IBM, USA). p < 0.05 was considered as statistically significant.