J Basic Clin Physiol Pharmacol 2014; aop
Adeolu Alex Adedapo*, Bisi Olajumoke Adeoye, Margaret Oluwatoyin Sofidiya and Ademola Adetokunbo Oyagbemi
Antioxidant, antinociceptive and anti-inflammatory properties of the aqueous and ethanolic leaf extracts of Andrographis paniculata in some laboratory animals Abstract Background: The study was designed to evaluate the anti-inflammatory, analgesic and antioxidant properties of Andrographis paniculata leaf extracts in laboratory animals. Methods: The dried and powdered leaves of the plant were subjected to phytochemical and proximate analyses. Its mineral content was also determined. Acute toxicity experiments were first performed to determine a safe dose level. The plant material was extracted using water and ethanol as solvents. These extracts were then used to test for the anti-inflammatory, analgesic and antioxidant properties of the plant. The anti-inflammatory tests included carrageenan-induced and histamine-induced paw oedema. The analgesic tests conducted were formalin paw lick test and acetic acid writhing test. The antioxidant activities of the extracts of A. paniculata were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), total polyphenol (TP) and 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) using ascorbic acid as standard for both DPPH and FRAP, and gallic acid as a standard for both TP and ABTS. Results: The acute toxicity experiment demonstrated that the plant is safe at high doses even at 1600 mg/kg. It was observed that the ethanolic extract of A. paniculata had
*Corresponding author: Adeolu Alex Adedapo, Faculty of Veterinary Medicine University of Ibadan, University of Ibadan – Veterinary Physiology, Biochemistry and Pharmacology, Oyo State 20005, Nigeria, Phone: +2348162746222, E-mail: [email protected] Bisi Olajumoke Adeoye: Department of Veterinary Physiology, Biochemistry and Pharmacology, University of Ibadan, Ibadan, Nigeria Margaret Oluwatoyin Sofidiya: Department of Pharmacognosy, University of Lagos, Lagos, Nigeria Ademola Adetokunbo Oyagbemi: Department of Veterinary Physiology, Biochemistry and Pharmacology, University of Ibadan, Ibadan, Nigeria
higher antioxidant activity than the aqueous extract. The experiments using both extracts may suggest that the extracts of A. paniculata leaves possess anti-inflammatory, analgesic and antioxidant properties, although the ethanolic extract seemed to have higher biological properties than the aqueous extract. Conclusions: The results from this study may have justified the plant’s folkloric use for medicinal purpose. Keywords: Andrographis paniculata; aqueous extract; anti-oxidant; anti-inflammatory; antinociceptive; ABTS; DPPH; ethanol extract; ferric reducing antioxidant power (FRAP). DOI 10.1515/jbcpp-2014-0051 Received April 17, 2014; accepted July 17, 2014
Introduction Andrographis paniculata (Burm.f.) Wall. ex Nees, commonly known as “king of bitters”, is an annual-branched, erect-running herb 0.5–1 m in height. The aerial parts of the plant (leaves and stems) are used to extract the active phytochemicals. It grows abundantly in south-eastern Asia – India (and Sri Lanka), Pakistan and Indonesia – but it is cultivated extensively in China and Thailand [1], the East and West Indies, and Mauritius [2]. Normally grown from seeds, Andrographis is ubiquitous in its native areas: it grows in pine, evergreen and deciduous forest areas, and along roads and in villages. Owing to its well-known medicinal properties, it is also cultivated very easily because it grows in all types of soil. The leaves contain the highest amount of andrographolide (2.39%), the most medicinally active phytochemical in the plant, whereas the seeds contain the lowest [3]. The primary medicinal component of Andrographis is andrographolide. It has a very bitter
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2 Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata
taste, has a colourless crystalline appearance, and is called a “diterpene lactone”, a chemical name that describes its ring-like structure. Other active components include 14-deoxy-11,12didehydroandrographolide, andrographosterin and stigmasterol, which was isolated from an astrographis preparation [4]. Andrographolides are excreted fairly rapidly from the body via the urine and gastrointestinal tract. The wide tissue and organ distribution and the immune-stimulating and regulatory actions of Andrographis paniculata (AP) make it an ideal candidate in the prevention and treatment of many diseases and conditions [5]. Andrographis paniculata is known to exhibit the following properties: analgesic [6], anti-inflammatory [7], antibacterial [2], antimalarial [8], antipyretic [9], antiviral [10], anticancer [11] and cardioprotective [12], among others. Antioxidants are agents needed to neutralize free radicals and are normally produced through exogenous and endogenous sources [13]. Herbs or spices prevent or inhibit the deleterious consequences of oxidative stress owing to the presence of natural antioxidants in them [14]. Any drug formulations that contain antioxidants are used in the prevention and treatment of complex diseases, such as atherosclerosis, stroke, diabetes, Alzheimer’s disease and cancer [14, 15]. Inflammation refers to a complex reaction to injury, irritation, or foreign invaders – collectively referred to as an “insult” – and is characterized by pain, swelling, redness and heat. It must be emphasized that whereas inflammation is a natural reaction to repair tissue damage or attack foreign invaders, the process often results in destruction of adjacent cells and/or tissues. Hence, there is overwhelming evidence that inflammation plays a critical role in many diseases, including asthma, multiple sclerosis, cardiovascular disease, Alzheimer’s disease, bowel disorders and cancer [16]. In the present study, the anti-oxidant, anti-inflammatory and analgesic potentials of the aqueous and ethanolic extracts of the leaves of the plant were evaluated to validate the folkloric claim to its medicinal use.
Plant collection and processing The leaves of A. paniculata were collected from the University of Ibadan Botanical Garden Ibadan, Nigeria in January 2013. The plants were identified and authenticated with voucher numbers: 2846 by the herbarium curator of the Department of Botany, University of Ibadan, Ibadan, Oyo State, Nigeria. The leaves were cleaned with distilled water and air dried in a well-ventilated shady room. The dried leaves were ground to powder using a blender. Some of the powdered leaves were measured separately for proximate and phytochemical analyses.
Leaf extraction Ethanolic extraction: The shade-dried leaves of the plant were extracted following the method of Mhadhebi et al. [17]. The ground powder was extracted in cold ethanol in a screw-capped flask and shaken at room temperature every 2 h for 48 h. The solvent was filtered, squeezed off and evaporated off under reduced pressure in a rotatory evaporator to obtain the crude extract. The resulting ethanol extract was then used for the experiments. Aqueous extraction: The procedure followed for aqueous extraction was the same as that for ethanolic extraction. The ground powder was extracted in distilled water in a screwcapped flask and shaken at room temperature every 2 h for 48 h. The resulting aqueous extract was then used for the experiments.
Experimental animals The laboratory animals used in this work were albino rats (Rattus norvegicus) (100–200 g) and mice (15–30 g) of both sexes. They were obtained from the Animal Holding Unit of the Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria. The animals were housed in clean metabolic cages, placed in well-ventilated house conditions (temperature: 28–31 °C; photoperiod: 12 h natural light and 12 h darkness; humidity: 50%–55%). They were also allowed free access to standard rat pellets and fresh water ad libitum. The cages were cleaned of waste once daily. All the animals were acclimatized to laboratory conditions for two weeks before commencement of experiments. All experimental protocols were conducted in compliance with the National Institute of Health Guide for Care and Use of Laboratory Animals.
Materials and methods Chemicals and reagents
Acute toxicity experiment
Chemicals used included carrageenan, acetic acid, formalin, Tween-80, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ascorbic acid, and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), which were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The standard drug used was indomethacin. All chemicals and drugs used were of analytical grade.
Following the method of Sawadogo et al. [18], the mice were randomly divided into six groups (A–F) with five animals in each group. Group A received distilled water (10 mL/kg) and served as the control, whereas groups B to F were administered with the aqueous extracts at 100, 200, 400, 800 and 1600 mg/kg, respectively. Monitoring of the parameters commenced immediately after oral administration of the extract for sign of acute toxicity, morbidity
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Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata 3
and mortality. The animals were observed at 0, 1, 2, 4, 6, 8, 12, 24 and 48 h.
as reference drug. The same procedure was repeated for the aqueous leaf extract of this plant.
Qualitative analysis: Qualitative analysis was performed using the ground (powdered) leaf of the plant. The constituents tested for included flavonoids, alkaloids, saponins, phenolic groups, cyanogenetic glycosides and antraquinones [19].
Anti-oxidant studies
Proximate analysis: Proximate analyses were conducted on the powdered ground leaves of the plant. Standard methods of analyses as described by Pearson were utilized in the present study for the determination of moisture content, total ash, protein content, lipid content and fiber content [20, 21].
Analgesic activities Acetic acid-induced writhing: The mice were divided into five groups (n = 4, each). Afterwards, they were pre-treated with the ethanolic extracts (50, 100 and 200 mg/kg, orally) and Tween 80 solution (0.2%, orally), whereas positive control was treated with indomethacin (10 mg/kg, orally) 30 min before an injection of 0.6% acetic acid (0.2 mL/animal, i.p.). Each animal was isolated in an individual observation chamber and 5 min after acetic acid injection, the cumulative number of writhing responses was recorded for 15 min [18]. The same procedure was repeated for the aqueous leaf extract of this plant. Formalin-induced test: The mice were divided into five groups (n = 4, each) and treated orally with vehicle (control), the ethanolic leaf extracts (50, 100 and 200 m/kg, orally) and indomethacin (10 mg/kg, orally). After 30 min, 0.1 mL of 2.5% formalin solution (0.92% formaldehyde in 0.9% saline) was injected into the subplantar area of the right hind paw. The duration of paw licking was measured at 0–10 min (first phase) and 20–30 min (second phase) after formalin administration [22]. The same procedure was repeated for the aqueous leaf extract of this plant.
Anti-inflammatory activity Carrageenan-induced paw edema in rats: Pedal inflammation in male albino rats was produced according to Guay et al. [23]. An injection of 0.1 mL of 1% carrageenan was delivered into the right hind foot of the rat under the subplantar aponeurosis. The rats were treated with the ethanolic leaf extracts (50, 100, 200 mg/kg, i.p) 60 min before carrageenan injection. The inflammation was quantified by measuring the volume displaced by the paw using a meter ruler and thread 0, 1, 2 and 3 h after carrageenan injection. Indomethacin (10 mg/kg) was given as reference drug. The same procedure was repeated for the aqueous leaf extract of this plant. Histamine-induced paw edema in rats: Pedal inflammation in male albino rats was produced according to Shim and Oh [24]. An injection of 0.1 mL of 1% histamine was delivered into the right hind foot of the rat under the subplantar aponeurosis. The rats were treated with the ethanolic leaf extracts (50, 100, 200 mg/kg, i.p.) 60 min before histamine injection. The inflammation was quantified by measuring the volume displaced by the paw using a meter ruler and thread 0, 1, 2 and 3 h after histamine injection. Indomethacin (10 mg/kg) was given
ABTS radical scavenging assay: The method described by Re et al. [25] was followed. The stock solution was prepared by mixing equal quantities of ABTS and potassium persulfate (K2S2O8) in 40 mL of distilled water. About 100 μL of this solution was added to 39 mL of methanol. About 200 μL of the plant extract (10 and 20 mg) were mixed with 2400 μL of ABTS, incubated for 15 min at room temperature and read with a spectrophotometer at 734 nm. Results were calculated and expressed in % and compared with gallic acid. DPPH radical scavenging assay: About 24 mg of DPPH was dissolved in 100 mL of methanol and stored at –20 °C as the stock solution. About 20 mL of stock was added to 90 mL of methanol and taken as the working solution. About 2850 μL of this working solution was mixed with 300 μL of extract (10 and 20 mg/mL), and then left for 30 min at room temperature. Absorbance of the mixture was measured using a spectrophotometer at 515 nm. Ascorbic acid was used as reference in accordance with the method of Liyana-Pathirana and Shahidi [26]. The ability to scavenge DPPH radical was calculated using the following equation based on the calibration curve: y = 1E–04x, R2 = 0.987, where x is the absorbance and y is the ascorbic acid equivalent.
Determination of total polyphenolics Total phenol contents were determined by the modified Folin-Ciocalteu method [27]. Folin was diluted with distilled water in the ratio 1:10, and 7.5 g of sodium bicarbonate was dissolved in 100 mL of distilled water. About 100 μL of the plant extract at 10 and 20 mg/kg were mixed with 0.5 mL of Folin; after 5 min, 0.4 mL of Na2CO3 was added. The tubes were allowed to stand for 2 h at room temperature for colour development. Absorbance was then measured spectrophotometrically at 760 nm. Results were expressed in μmol/L and compared with gallic acid. Ferrous reducing activity power (FRAP) assay: For this assay, the method by Benzie and Strain [28] was adopted with slight modifications. The stock solutions included 300 mM acetate buffer (prepared), pH 3.6, 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl3·6H20 solution. The stock solution was prepared by mixing 225 mL acetate buffer, 0.625 g of TPTZ in 20 mL of (0.3 mL of HCl in 250 mL of distilled water), 0.108 g of FeCl3 in 20 mL of (0.3 mL of HCl in 250 mL of distilled water). About 100 μL of the plant extract (10 and 20 mg/kg) were allowed to react with 300 μL of the FRAP solution and incubated at 37 °C in the oven for 30 min. Readings were taken using a spectrophotometer at 593 nm. Results were calculated and expressed in μmol/L and compared with ascorbic acid.
Statistical analysis The data generated were presented as mean ± SD. Statistical analysis was performed by using GraphPad Prism 5. The results were further
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4 Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata subjected to one-way ANOVA, the variance means were separated using the Student’s t-test and differences between means were considered significant at p < 0.05.
Table 2 Proximate analysis result of the plant A. paniculata. Carbohydrate, %
Protein, %
Crude fat, %
Moisture, %
Ash, %
Crude fibre, %
60.20
3.72
2.93
3.00
2.71
27.46
Results Qualitative analysis of the powdered leaves of the plant showed that it is rich in reducing sugars, saponins, flavonoids, phenolic groups, and anthraquinone. The study also showed that cyanogenetic glycosides and alkaloids are absent (Table 1). The quantitative estimation of the percentage proximate compositions of A. paniculata is shown in Table 2. It was revealed that the plant contains carbohydrate (60.20%), protein (3.72%), crude fat (2.93%), moisture (3.00%), ash (2.71%) and crude fiber (27.46%). The analysis also showed that the plant contains sodium, potassium, magnesium and calcium in large amounts. It also has zinc, iron, copper, cobalt, chromium, manganese, lead and nickel in trace amounts (Table 3). The acute toxicity experiments of the aqueous and ethanolic crude extracts of the plant showed that doses of 100, 200, 400, 800 and 1600 mg/kg were tolerated by animals. There were no toxic effects observed because the animals were asymptomatic. There was no death and apparent behavioural changes recorded during the study in all treatment groups. The effect of indomethacin (10 mg/kg) and aqueous extract (50 mg and100 mg/kg) on carrageenan-induced paw oedema was more pronounced 3 h after carrageenan injection, whereas the effect of the extract (200 mg/kg) was more pronounced 1 h after the injection. The effect of the extract (200 mg/kg) was higher 1, 2 and 3 h after
Table 1 Qualitative analysis result of the plant A. paniculata. Reducing sugar Saponins Flavonoids Phenolic group Cyanogenetic glycosides Alkaloids Anthraquinone
+++ ++ ++ ++ – – ++
the injection compared to that of the reference drug (indomethacin). The effect of the extract at 100 mg/kg and the reference drug (indomethacin) was the same 2 h after the injection. In the case of the ethanolic extract, the effect of 50, 100 and 200 mg/kg doses and indomethacin (10 mg/kg) on carrageenan-induced paw edema were most pronounced 3 h after the injection. The effect of 200 mg/kg of the extract on carrageenan-induced paw edema showed greater anti-edema effect relative to the reference drug (indomethacin). The ethanolic extract also appeared to have a higher anti-edema effect than its aqueous counterpart (Table 4). The effect of the aqueous extract (50 and 200 mg) on histamine-induced paw edema was more pronounced 2 h after histamine injection, whereas that of the extract (100 mg) and indomethacin (10 mg/kg) was more pronounced 3 h after the injection. In the case of the ethanolic extract, the three doses and the reference drug (indomethacin, 10 mg/kg) produced the most pronounced effect 3 h after carrageenan injection. The effect of 200 mg/kg of the extract on histamine-induced paw edema showed greater anti-oedema effect relative to indomethacin (Table 5). The aqueous extract caused a decrease in the number of writhes at doses of 50, 100 and 200 mg/kg when compared to the control. The 200 mg/kg dose of the extract and indomethacin demonstrated a high antinociceptive power at 72.8% and 88.4%, respectively. The three doses of the ethanolic extract also caused a decrease in the number of writhes when compared with the control. However, the 200 mg/kg dose of this extract showed antinociceptive power of 52.3%, whereas that of indomethacin was 85.8%. The aqueous extract seemed to have a higher antinociceptive effect when compared with the ethanolic extract (Table 6). Table 7 shows the effect of the aqueous extract of AP and indomethacin on the paw lick test on mouse induced by formalin. The extract exhibited dose-dependent inhibition of paw lick by mice especially in the early phase. However, the effect of indomethacin was higher than that
Table 3 Mineral contents (mg/100 g) of the plant A. paniculata. Na
K
Mg
Ca
Zn
Fe
Cu
Co
Cr
Mn
Pb
Cd
Ni
59.45
58.2
37.7
149.5
6.05
4.35
2.25
1.05
0.55
1.6
0.05
Nil
0.55
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Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata 5
Table 4 Anti-inflammatory activity of the aqueous and ethanolic leaf extracts of A. paniculata and indomethacin on histamine-induced oedema in the right hind paw of rats. Treatment
Aqueous extract Ethanolic extract
Time, h 0 1 2 3 0 1 2 3
Extract dose, mg/kg; indomethacin, 10 mg/kg 50
100
200
Indomethacin
23.4 ± 1.5 26.4 ± 1.3 (–0.8) 25.6 ± 0.9 (8.7) 24.8 ± 1.1(6.70) 22.6 ± 0.5 26.0 ± 0.7 (2.3) 25.8 ± 0.8 (4.4) 24.4 ± 0.9 (7.6)
21.6 ± 0.5 24.0 ± 0.7 (8.4) 23.4 ± 1.1 (7.3) 22.6 ± 0.9 (15) 21.8 ± 0.4 25.2 ± 0.4 (5.3) 24.2 ± 0.8 (10.4) 23.0 ± 0.7 (12.9)
23.0 ± 1.2 24.2 ± 1.1 (7.6) 23.4 ± 0.9 (15.2) 23.4 ± 1.5 (12) 22.0 ± 1.7 24.2 ± 1.6 (9.0) 23.8 ± 1.9 (11.9) 22.2 ± 1.6 (15.9)
22.4 ± 0.5 25.4 ± 0.9 (3) 25.2 ± 0.8 (8.7) 24.2 ± 1.3 (9) 22.2 ± 0.4 25.2 ± 0.8 (5.3) 24.2 ± 1.3 (10.4) 23.6 ± 1.5 (10.6)
Control (2 mL/kg) 23.2 ± 1.1 26.2 ± 0.8 27.6 ± 1.5 26.6 ± 0.9 22.6 ± 0.9 26.6 ± 1.3 27 ± 1.4 26.4 ± 1.5
(n = 4), mean ± SD. Table 5 Anti-inflammatory activity of the aqueous and ethanolic leaf extracts of A. paniculata and indomethacin on carrageenan-induced edema in the right hind paw of rats. Treatment
Aqueous extract Ethanolic extract
Time, h 0 1 2 3 0 1 2 3
Extract dose, mg/kg; indomethacin, 10 mg/kg 50
100
200
24.4 ± 0.5 28.2 ± 0.8 (0.7) 27.2 ± 0.4 (2.1) 25.0 ± 1.0 (8.8) 24.4 ± 0.5 28.2 ± 0.8 (–0.7) 27.2 ± 0.4 (2.8) 25.0 ± 1.0 (11.4)
22.6 ± 1.8 25.6 ± 1.3 (9.9) 24.0 ± 1.4 (2.1) 22.8 ± 1.1 (16.8) 22.6 ± 1.8 25.6 ± 1.3 (8.6) 24.0 ± 1.4 (14.3) 22.8 ± 1.1 (19.2)
22.4 ± 1.1 23.0 ± 1.7 (19) 22.6 ± 1.1 (18.7) 22.4 ± 1.1 (18.3) 22.4 ± 1.1 23.0 ± 1.6 (17.9) 22.6 ± 1.1 (19.3) 22.4 ± 1.1 (20.6)
Control (2 mL/kg)
22.4 ± 0.9 28.4 ± 0.7 27.8 ± 1.6 27.4 ± 1.7 22.4 ± 0.5 28.0 ± 1.0 28.0 ± 1.4 28.2 ± 1.6
Indomethacin
22.8 ± 0.8 25.2 ± 1.5 (10) 24.0 ± 1.2 (14) 23.6 ± 1.1 (14) 23.0 ± 0.7 24.6 ± 0.5 (12.1) 23.8 ± 0.8 (15) 23.4 ± 1.1 (17)
(n = 4), mean ± SD.
of the highest dose of the extract. In contrast, the opposite was true between the reference drug and the 200 mg/kg dose of the extract in the late phase. The effect of the ethanolic extract on the paw lick test on mouse was comparable to that of the aqueous extract, except that the aqueous extract had a slightly higher antinociceptive activity than the ethanolic extract. Also in the late phase, the 200 mg/kg dose of the ethanolic extract showed a more antinociceptive effect than indomethacin (Table 8). In the case of the antioxidant activities of both extracts, the ethanolic extract showed a higher effect compared with the aqueous extract (Table 9).
Discussion The result of the acute toxicity experiments showed the absence of lethality or toxic side effects after oral administration of both aqueous and ethanolic extracts of A. paniculata even at the high dose of 1600 mg/kg. This result might be an indication of the non-toxic nature of the plant. In the sub-acute toxicity experiments performed on the aqueous crude extract of this plant in rats, it was found that the aqueous crude extract did not cause any significant changes in the red blood cell parameters and indices, although it caused significant changes in
Table 6 Analgesic effect of aqueous and ethanolic leaf extracts of A. paniculata and indomethacin on mouse writhing reflex induced by acetic acid. Treatment
Aqueous extract Ethanolic extract
50
100
16.0 ± 5.8 (68.9) 33.0 ± 6.2 (33.0)
16.0 ± 7.2 (68.9) 29.8 ± 6.2 (39.6)
Extract dose, mg/kg; indomethacin, 10 mg/kg 200 Indomethacin 14.0 ± 1.8 (72.8) 23.5 ± 3.7 (52.3)
(n = 4), mean ± SD. Percentage inhibition of writhes in parentheses.
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6.0 ± 4.1 (88.4) 7.0 ± 3.2 (85.8)
Control (2 mL/kg) 51.5 ± 14.1 (0) 49.3 ± 13.3 (0)
6 Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata Table 7 Analgesic effect of aqueous leaf extract of A. paniculata and indomethacin on paw lick test on mouse induced by formalin. Parameters
Control
52.5 ± 2.5 19.5 ± 0.6
Early phase Late phase
Indomethacin
9.8 ± 3.0 (81.3) 14.0 ± 8.5 (28.2)
Extract 50 mg
21.8 ± 1.7 (58.5) 17.0 ± 0.8 (12.8)
100 mg
20.3 ± 6.5 (61.3) 14.8 ± 3.8 (24.1)
200 mg
14.8 ± 4.1 (71.8) 6.3 ± 2.6 (67.7)
The extract (50, 100 and 200 mg/kg) and indomethacin exhibited higher inhibition at the early phase than at the late phase.
Table 8 Analgesic effect of ethanolic leaf extracts of A. paniculata and indomethacin on paw lick test on mouse induced by formalin. Parameters
Control
51.0 ± 1.8 19.8 ± 1.0
Early phase Late phase
Indomethacin
Extract
9.8 ± 3.0 (80.8) 14.0 ± 8.5 (29.3)
50 mg
100 mg
200 mg
25.0 ± 2.2 (51.0) 19.5 ± 1.7 (1.5)
29.3 ± 1.0 (42.6) 14.5 ± 5.4 (26.8)
14.0 ± 2.4 (72.6) 7.8 ± 3.8 (44.3)
(n = 4), mean ± SD. The extract (50, 100 and 200 mg/kg) and indomethacin exhibited high inhibition at the early phase.
white blood cell count and its differentials. The extract also caused significant changes in the levels of alkaline phosphatase, aspartate amino transferase, gamma-glutamyl transferase and alanine amino transferase. Increases were also observed in the levels of total protein, globulin, total bilirubin and unconjugated bilirubin. Histologically, the liver of the animals in some groups showed moderate vacuolar degeneration and necrosis of hepatocytes, whereas the kidney also showed eosinophilic casts in the tubules [29]. Thus, caution should be exercised in using this plant for medicinal purpose especially for prolonged periods. Inflammation induced by carrageenan is an acute one and it involves the synthesis or release of mediators, such as prostaglandins, serotonin, histamine, etc., at the injured site, and these mediators all cause pain and fever [30]. Anti-inflammatory drugs act by inhibiting these mediators from getting to the injured site, thereby ameliorating pain and inflammation. The highest antiinflammatory effect on carrageenan-induced edema was demonstrated by the 200 mg/kg dose of ethanolic extract 3 h after carrageenan injection. The extracts reduced the
oedema produced by histamine, suggesting that the antiinflammatory activity of the extracts is probably due to its anti-histamine activity. Histamine is a potent vasodilator that increases vascular permeability [31]. Therefore, the extracts could have acted either by inhibiting the synthesis of these inflammatory mediators or even their release from the storage sites. The results showed that all the extracts used in this study were effective. The anti-inflammatory and analgesic effects are comparable to that of indomethacin, the standard anti-inflammatory drug used. Phytochemical analysis of A. paniculata shows the presence of reducing sugars, saponin, phenolic group and alkaloids. The various phytochemical compounds detected are known to have beneficial importance in medicinal science. Phenols are said to offer resistance to diseases and accounts for most of the anti-oxidant activity in plants [32]. Flavonoids show anti-allergic, anti-inflammatory, anti-microbial and anti-cancer activities [33]. Alkaloids have been used to treat diseases such as malaria, and glycosides serve as defence mechanisms against many microorganisms. Saponin protects
Table 9 DPPH and ABTS radical scavenging activities, as well as FRAP and TP contents, of both aqueous and ethanolic leaf extract of A. paniculata.
DPPH (AAE)
FRAP (AAE)
TP (GAE)
ABTS (GAE)
Aqueous extract Ethanol extract
9.28 ± 0.002 (11.96%) 12.91 ± 0.007a (76.6%)
5.00 ± 0.47 (9.8%) 50.37 ± 0.23a (42%)
42.95 ± 0.02 (6.1%) 78.56 ± 0.04a (58.4%)
1.35 ± 0.07 (30%) 6.21 ± 0.003a (82.7%)
p < 0.001.
a
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Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata 7
the plant against microbes and fungi. The plant is also known to contain carbohydrate, protein, crude fat, ash and crude fibre. The crude fibre content of the plant is high, and adequate intake of dietary fibre can lower the serum cholesterol level, risk of coronary heart disease, hypertension, constipation, diabetes, colon and breast cancer [34]. The relatively high level of carbohydrate, protein and others in this plant is attributed to its nutrient values. Analysis of the mineral content of this plant showed that it is rich in sodium, potassium, magnesium, calcium, zinc, iron, copper, cobalt, chromium, manganese, lead and nickel. Iron is an essential trace element for haemoglobin formation, normal functioning of the central nervous system and in the oxidation of fats, carbohydrates and protein. Therefore, consumption of the plant will increase the level of iron in the body. Although iron is an essential nutrient for plants, its accumulation within cells can be toxic. Thus, plants respond to both iron deficiency and iron excess by inducing expression of different gene sets [35]. Copper is naturally present in fruits and vegetables and is necessary for the proper growth and functioning of plants. The primary source of this mineral is from the soil, but small amounts may result from copper-based fungicides. Although copper concentrations in plants tend to increase with increasing copper concentrations in the soil, soil properties such as the acidity level and organic matter content can affect the amount of copper taken up [36]. Although the radical scavenging abilities of the extracts were less than those of ascorbic acid (85.3%) and gallic acid, the study showed that the extracts have proton-donating ability and could serve as free radical inhibitors or scavengers, acting possibly as a primary antioxidant. The ethanol extract particularly showed great promise with respect to free radical scavenging activity. Various phytochemical compounds detected from the plant are known to have beneficial importance in medicinal science [37]. Hence, the observed antioxidant activities may be due to the presence of these phytochemical compounds and nutritive values. Thus, the findings support the reported therapeutic use of this plant in traditional medicine for the alleviation of pain and inflammation [38]. Therefore, it could be concluded that both the aqueous and ethanolic extracts of A. paniculata have great medicinal potential, and this study may have justified the folkloric use of this plant for this purpose. Acknowledgments: This study was conducted with a research grant (SRG/FVM/2010A) from the University of Ibadan given to Dr. Adeolu Adedapo.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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8 Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata 14. Khalaf NA, Shakya AK, Al-Othman A, El-Agbar Z, Farah H. Antioxidant activity of some common plants. Turk J Biol 2008;32:51–5. 15. Devasagayam TP, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS, Lele RD. Free radical and antioxidants in human health. J Assoc Physicians India 2004;53:794–804. 16. Ganey PE, Luyendyk JP, Maddox JF, Roth RA. Adverse hepatic drug reactions: inflammatory episodes as consequence and contributor. Chem Biol Interact 2004;150:35–51. 17. Mhadhebi L, Mhadhebi A, Robert J, Bouraoui A. Antioxidant, anti-inflammatory and antiproliferative effects of aqueous extracts of three Mediterranean brown seaweeds of the genus Cystoseira. Iran J Pharmacol Res 2014;13:207–220. 18. Sawadogo WR, Boly R, Lompo MS. Anti-inflammatory, analgesic and antipyretic activities of Dicliptera verticillata. Int J Pharmacol 2006;2:435–8. 19. Moody JO, Robert VA, Connolly JD, Houghton PJ. Anti-inflammatory activities of the methanol extracts and isolated furanoditerpene constituent of Sphenenocentrum jollyanum Pierre (Meispermaceae). J Ethnopharmacol 2006;104:87–91. 20. Pearson D. Chemical analysis of foods, 7th ed. Churchill Livingstone: London, 1976:218–336. 21. AOAC. Association of Official Analytical Chemists International (AOAC) Official Methods of Analysis, 15th ed. AOAC International, Gaithersburg, Maryland, USA. AOAC 1990;1:644, 2:993. 22. Ceccarelli I, Fiorenzani P, Massafra C, Aloisi AM. Long-term ovariectomy changes formalin-induced licking in female rats: The role of estrogens. Reprod Biol Endocrin 2003;1:24. 23. Guay J, Bateman K, Gordon R, Mancini J, Riendeau D. Carrageenan-induced paw edema in rat elicits a predominant prostaglandin E2 (PGE2) response in the central nervous system associated with the induction of microsomal PGE2 synthase-1. J Biol Chem 2004;279:24866–72. 24. Shim W, Oh U. Histamine-induced itch and its relationship with pain. Mol Pain 2008;4:29. 25. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Bio Med 1999;26:1231–7. 26. Liyana-Pathiranan CM, Shahidi F. Antioxidant activity of commercial soft and hard wheat (Triticum aestivum L.)
as affected by gastric pH conditions. J Agr Food Chem 2005;53:2433–40. 27. Wolfe K, Wu X, Liu RH. Antioxidant activity of apple peels. J Agr Food Chem 2003;51:609–14. 28. Benzie IFF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”. The FRAP Assay. Anal Biochem 1996;238:70–6. 29. Adedapo AA, Obadan AY, Ohore OG. Some toxicological effects of the aqueous leaf extract of Andrographis paniculata in rats. Trop Vet 2009;27:1–9. 30. Asongalem E, Foyer H, Ekoo S, Dimo T, Kamtchouig P. Antiinflammatory, lack of central analgesia and antipyretic properties of Acanthus montanus (Nees) T. Anderson. J Ethnopharmacol 2004;95:63–8. 31. Linardi A, Costa SK, DeSilva GR, Antunes E. Involvement of Kinins, mast cells and sensory neurons in the plasma exudation and paw oedema induced by staphylococcal enterotoxin B in the mouse. Eur J Pharmacol 2002;399:325–42. 32. Aliyu AB, Musa AM, Sallau MS, Oyewale AO. Proximate composition, mineral elements and anti-nutritional factors of Anisopus mannii N. E. Br. (Asclepiadaceae). Trends Appl Sc Res 2009;4:68–72. 33. Aiyelaagbe OO, Osamudiamen PM. Phytochemical screening for active compounds in Mangifera indica. Plant Sci Res 2009;2:11–3. 34. Ishida H, Suzuno H, Sugiyama N, Innami S, Todokoro T, Maekawa A. Nutritional evaluation of chemical component of leaves, stalks and stems of sweet potatoes (Ipomea batatas Poir.). Food Chem 2000;68:359–67. 35. Wei J, Theil EC. Identification and characterization of the iron regulatory element in the ferritin gene of a plant (soybean). J Biol Chem 2000;275:17488–93. 36. Guideline for Use at Contaminated Sites in Ontario (revised 1997). Ontario Ministry of the Environment, Queens Park, Toronto, Ontario. 37. Lee DH, Folsom AR, Harnack L. Does supplemental vitamin C increase cardiovascular disease risk in women with diabetes? Am J Clin Nutr 2004;80:1194–2000. 38. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian medicinal plants, 1st ed. Publication and Information Directorate, CSIR, New Delhi, 1956.
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Adeolu Alex Adedapo*, Bisi Olajumoke Adeoye, Margaret Oluwatoyin Sofidiya and Ademola Adetokunbo Oyagbemi
Antioxidant, antinociceptive and anti-inflammatory properties of the aqueous and ethanolic leaf extracts of Andrographis paniculata in some laboratory animals Abstract Background: The study was designed to evaluate the anti-inflammatory, analgesic and antioxidant properties of Andrographis paniculata leaf extracts in laboratory animals. Methods: The dried and powdered leaves of the plant were subjected to phytochemical and proximate analyses. Its mineral content was also determined. Acute toxicity experiments were first performed to determine a safe dose level. The plant material was extracted using water and ethanol as solvents. These extracts were then used to test for the anti-inflammatory, analgesic and antioxidant properties of the plant. The anti-inflammatory tests included carrageenan-induced and histamine-induced paw oedema. The analgesic tests conducted were formalin paw lick test and acetic acid writhing test. The antioxidant activities of the extracts of A. paniculata were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), total polyphenol (TP) and 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) using ascorbic acid as standard for both DPPH and FRAP, and gallic acid as a standard for both TP and ABTS. Results: The acute toxicity experiment demonstrated that the plant is safe at high doses even at 1600 mg/kg. It was observed that the ethanolic extract of A. paniculata had
*Corresponding author: Adeolu Alex Adedapo, Faculty of Veterinary Medicine University of Ibadan, University of Ibadan – Veterinary Physiology, Biochemistry and Pharmacology, Oyo State 20005, Nigeria, Phone: +2348162746222, E-mail: [email protected] Bisi Olajumoke Adeoye: Department of Veterinary Physiology, Biochemistry and Pharmacology, University of Ibadan, Ibadan, Nigeria Margaret Oluwatoyin Sofidiya: Department of Pharmacognosy, University of Lagos, Lagos, Nigeria Ademola Adetokunbo Oyagbemi: Department of Veterinary Physiology, Biochemistry and Pharmacology, University of Ibadan, Ibadan, Nigeria
higher antioxidant activity than the aqueous extract. The experiments using both extracts may suggest that the extracts of A. paniculata leaves possess anti-inflammatory, analgesic and antioxidant properties, although the ethanolic extract seemed to have higher biological properties than the aqueous extract. Conclusions: The results from this study may have justified the plant’s folkloric use for medicinal purpose. Keywords: Andrographis paniculata; aqueous extract; anti-oxidant; anti-inflammatory; antinociceptive; ABTS; DPPH; ethanol extract; ferric reducing antioxidant power (FRAP). DOI 10.1515/jbcpp-2014-0051 Received April 17, 2014; accepted July 17, 2014
Introduction Andrographis paniculata (Burm.f.) Wall. ex Nees, commonly known as “king of bitters”, is an annual-branched, erect-running herb 0.5–1 m in height. The aerial parts of the plant (leaves and stems) are used to extract the active phytochemicals. It grows abundantly in south-eastern Asia – India (and Sri Lanka), Pakistan and Indonesia – but it is cultivated extensively in China and Thailand [1], the East and West Indies, and Mauritius [2]. Normally grown from seeds, Andrographis is ubiquitous in its native areas: it grows in pine, evergreen and deciduous forest areas, and along roads and in villages. Owing to its well-known medicinal properties, it is also cultivated very easily because it grows in all types of soil. The leaves contain the highest amount of andrographolide (2.39%), the most medicinally active phytochemical in the plant, whereas the seeds contain the lowest [3]. The primary medicinal component of Andrographis is andrographolide. It has a very bitter
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2 Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata
taste, has a colourless crystalline appearance, and is called a “diterpene lactone”, a chemical name that describes its ring-like structure. Other active components include 14-deoxy-11,12didehydroandrographolide, andrographosterin and stigmasterol, which was isolated from an astrographis preparation [4]. Andrographolides are excreted fairly rapidly from the body via the urine and gastrointestinal tract. The wide tissue and organ distribution and the immune-stimulating and regulatory actions of Andrographis paniculata (AP) make it an ideal candidate in the prevention and treatment of many diseases and conditions [5]. Andrographis paniculata is known to exhibit the following properties: analgesic [6], anti-inflammatory [7], antibacterial [2], antimalarial [8], antipyretic [9], antiviral [10], anticancer [11] and cardioprotective [12], among others. Antioxidants are agents needed to neutralize free radicals and are normally produced through exogenous and endogenous sources [13]. Herbs or spices prevent or inhibit the deleterious consequences of oxidative stress owing to the presence of natural antioxidants in them [14]. Any drug formulations that contain antioxidants are used in the prevention and treatment of complex diseases, such as atherosclerosis, stroke, diabetes, Alzheimer’s disease and cancer [14, 15]. Inflammation refers to a complex reaction to injury, irritation, or foreign invaders – collectively referred to as an “insult” – and is characterized by pain, swelling, redness and heat. It must be emphasized that whereas inflammation is a natural reaction to repair tissue damage or attack foreign invaders, the process often results in destruction of adjacent cells and/or tissues. Hence, there is overwhelming evidence that inflammation plays a critical role in many diseases, including asthma, multiple sclerosis, cardiovascular disease, Alzheimer’s disease, bowel disorders and cancer [16]. In the present study, the anti-oxidant, anti-inflammatory and analgesic potentials of the aqueous and ethanolic extracts of the leaves of the plant were evaluated to validate the folkloric claim to its medicinal use.
Plant collection and processing The leaves of A. paniculata were collected from the University of Ibadan Botanical Garden Ibadan, Nigeria in January 2013. The plants were identified and authenticated with voucher numbers: 2846 by the herbarium curator of the Department of Botany, University of Ibadan, Ibadan, Oyo State, Nigeria. The leaves were cleaned with distilled water and air dried in a well-ventilated shady room. The dried leaves were ground to powder using a blender. Some of the powdered leaves were measured separately for proximate and phytochemical analyses.
Leaf extraction Ethanolic extraction: The shade-dried leaves of the plant were extracted following the method of Mhadhebi et al. [17]. The ground powder was extracted in cold ethanol in a screw-capped flask and shaken at room temperature every 2 h for 48 h. The solvent was filtered, squeezed off and evaporated off under reduced pressure in a rotatory evaporator to obtain the crude extract. The resulting ethanol extract was then used for the experiments. Aqueous extraction: The procedure followed for aqueous extraction was the same as that for ethanolic extraction. The ground powder was extracted in distilled water in a screwcapped flask and shaken at room temperature every 2 h for 48 h. The resulting aqueous extract was then used for the experiments.
Experimental animals The laboratory animals used in this work were albino rats (Rattus norvegicus) (100–200 g) and mice (15–30 g) of both sexes. They were obtained from the Animal Holding Unit of the Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria. The animals were housed in clean metabolic cages, placed in well-ventilated house conditions (temperature: 28–31 °C; photoperiod: 12 h natural light and 12 h darkness; humidity: 50%–55%). They were also allowed free access to standard rat pellets and fresh water ad libitum. The cages were cleaned of waste once daily. All the animals were acclimatized to laboratory conditions for two weeks before commencement of experiments. All experimental protocols were conducted in compliance with the National Institute of Health Guide for Care and Use of Laboratory Animals.
Materials and methods Chemicals and reagents
Acute toxicity experiment
Chemicals used included carrageenan, acetic acid, formalin, Tween-80, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ascorbic acid, and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), which were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The standard drug used was indomethacin. All chemicals and drugs used were of analytical grade.
Following the method of Sawadogo et al. [18], the mice were randomly divided into six groups (A–F) with five animals in each group. Group A received distilled water (10 mL/kg) and served as the control, whereas groups B to F were administered with the aqueous extracts at 100, 200, 400, 800 and 1600 mg/kg, respectively. Monitoring of the parameters commenced immediately after oral administration of the extract for sign of acute toxicity, morbidity
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Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata 3
and mortality. The animals were observed at 0, 1, 2, 4, 6, 8, 12, 24 and 48 h.
as reference drug. The same procedure was repeated for the aqueous leaf extract of this plant.
Qualitative analysis: Qualitative analysis was performed using the ground (powdered) leaf of the plant. The constituents tested for included flavonoids, alkaloids, saponins, phenolic groups, cyanogenetic glycosides and antraquinones [19].
Anti-oxidant studies
Proximate analysis: Proximate analyses were conducted on the powdered ground leaves of the plant. Standard methods of analyses as described by Pearson were utilized in the present study for the determination of moisture content, total ash, protein content, lipid content and fiber content [20, 21].
Analgesic activities Acetic acid-induced writhing: The mice were divided into five groups (n = 4, each). Afterwards, they were pre-treated with the ethanolic extracts (50, 100 and 200 mg/kg, orally) and Tween 80 solution (0.2%, orally), whereas positive control was treated with indomethacin (10 mg/kg, orally) 30 min before an injection of 0.6% acetic acid (0.2 mL/animal, i.p.). Each animal was isolated in an individual observation chamber and 5 min after acetic acid injection, the cumulative number of writhing responses was recorded for 15 min [18]. The same procedure was repeated for the aqueous leaf extract of this plant. Formalin-induced test: The mice were divided into five groups (n = 4, each) and treated orally with vehicle (control), the ethanolic leaf extracts (50, 100 and 200 m/kg, orally) and indomethacin (10 mg/kg, orally). After 30 min, 0.1 mL of 2.5% formalin solution (0.92% formaldehyde in 0.9% saline) was injected into the subplantar area of the right hind paw. The duration of paw licking was measured at 0–10 min (first phase) and 20–30 min (second phase) after formalin administration [22]. The same procedure was repeated for the aqueous leaf extract of this plant.
Anti-inflammatory activity Carrageenan-induced paw edema in rats: Pedal inflammation in male albino rats was produced according to Guay et al. [23]. An injection of 0.1 mL of 1% carrageenan was delivered into the right hind foot of the rat under the subplantar aponeurosis. The rats were treated with the ethanolic leaf extracts (50, 100, 200 mg/kg, i.p) 60 min before carrageenan injection. The inflammation was quantified by measuring the volume displaced by the paw using a meter ruler and thread 0, 1, 2 and 3 h after carrageenan injection. Indomethacin (10 mg/kg) was given as reference drug. The same procedure was repeated for the aqueous leaf extract of this plant. Histamine-induced paw edema in rats: Pedal inflammation in male albino rats was produced according to Shim and Oh [24]. An injection of 0.1 mL of 1% histamine was delivered into the right hind foot of the rat under the subplantar aponeurosis. The rats were treated with the ethanolic leaf extracts (50, 100, 200 mg/kg, i.p.) 60 min before histamine injection. The inflammation was quantified by measuring the volume displaced by the paw using a meter ruler and thread 0, 1, 2 and 3 h after histamine injection. Indomethacin (10 mg/kg) was given
ABTS radical scavenging assay: The method described by Re et al. [25] was followed. The stock solution was prepared by mixing equal quantities of ABTS and potassium persulfate (K2S2O8) in 40 mL of distilled water. About 100 μL of this solution was added to 39 mL of methanol. About 200 μL of the plant extract (10 and 20 mg) were mixed with 2400 μL of ABTS, incubated for 15 min at room temperature and read with a spectrophotometer at 734 nm. Results were calculated and expressed in % and compared with gallic acid. DPPH radical scavenging assay: About 24 mg of DPPH was dissolved in 100 mL of methanol and stored at –20 °C as the stock solution. About 20 mL of stock was added to 90 mL of methanol and taken as the working solution. About 2850 μL of this working solution was mixed with 300 μL of extract (10 and 20 mg/mL), and then left for 30 min at room temperature. Absorbance of the mixture was measured using a spectrophotometer at 515 nm. Ascorbic acid was used as reference in accordance with the method of Liyana-Pathirana and Shahidi [26]. The ability to scavenge DPPH radical was calculated using the following equation based on the calibration curve: y = 1E–04x, R2 = 0.987, where x is the absorbance and y is the ascorbic acid equivalent.
Determination of total polyphenolics Total phenol contents were determined by the modified Folin-Ciocalteu method [27]. Folin was diluted with distilled water in the ratio 1:10, and 7.5 g of sodium bicarbonate was dissolved in 100 mL of distilled water. About 100 μL of the plant extract at 10 and 20 mg/kg were mixed with 0.5 mL of Folin; after 5 min, 0.4 mL of Na2CO3 was added. The tubes were allowed to stand for 2 h at room temperature for colour development. Absorbance was then measured spectrophotometrically at 760 nm. Results were expressed in μmol/L and compared with gallic acid. Ferrous reducing activity power (FRAP) assay: For this assay, the method by Benzie and Strain [28] was adopted with slight modifications. The stock solutions included 300 mM acetate buffer (prepared), pH 3.6, 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl3·6H20 solution. The stock solution was prepared by mixing 225 mL acetate buffer, 0.625 g of TPTZ in 20 mL of (0.3 mL of HCl in 250 mL of distilled water), 0.108 g of FeCl3 in 20 mL of (0.3 mL of HCl in 250 mL of distilled water). About 100 μL of the plant extract (10 and 20 mg/kg) were allowed to react with 300 μL of the FRAP solution and incubated at 37 °C in the oven for 30 min. Readings were taken using a spectrophotometer at 593 nm. Results were calculated and expressed in μmol/L and compared with ascorbic acid.
Statistical analysis The data generated were presented as mean ± SD. Statistical analysis was performed by using GraphPad Prism 5. The results were further
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4 Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata subjected to one-way ANOVA, the variance means were separated using the Student’s t-test and differences between means were considered significant at p < 0.05.
Table 2 Proximate analysis result of the plant A. paniculata. Carbohydrate, %
Protein, %
Crude fat, %
Moisture, %
Ash, %
Crude fibre, %
60.20
3.72
2.93
3.00
2.71
27.46
Results Qualitative analysis of the powdered leaves of the plant showed that it is rich in reducing sugars, saponins, flavonoids, phenolic groups, and anthraquinone. The study also showed that cyanogenetic glycosides and alkaloids are absent (Table 1). The quantitative estimation of the percentage proximate compositions of A. paniculata is shown in Table 2. It was revealed that the plant contains carbohydrate (60.20%), protein (3.72%), crude fat (2.93%), moisture (3.00%), ash (2.71%) and crude fiber (27.46%). The analysis also showed that the plant contains sodium, potassium, magnesium and calcium in large amounts. It also has zinc, iron, copper, cobalt, chromium, manganese, lead and nickel in trace amounts (Table 3). The acute toxicity experiments of the aqueous and ethanolic crude extracts of the plant showed that doses of 100, 200, 400, 800 and 1600 mg/kg were tolerated by animals. There were no toxic effects observed because the animals were asymptomatic. There was no death and apparent behavioural changes recorded during the study in all treatment groups. The effect of indomethacin (10 mg/kg) and aqueous extract (50 mg and100 mg/kg) on carrageenan-induced paw oedema was more pronounced 3 h after carrageenan injection, whereas the effect of the extract (200 mg/kg) was more pronounced 1 h after the injection. The effect of the extract (200 mg/kg) was higher 1, 2 and 3 h after
Table 1 Qualitative analysis result of the plant A. paniculata. Reducing sugar Saponins Flavonoids Phenolic group Cyanogenetic glycosides Alkaloids Anthraquinone
+++ ++ ++ ++ – – ++
the injection compared to that of the reference drug (indomethacin). The effect of the extract at 100 mg/kg and the reference drug (indomethacin) was the same 2 h after the injection. In the case of the ethanolic extract, the effect of 50, 100 and 200 mg/kg doses and indomethacin (10 mg/kg) on carrageenan-induced paw edema were most pronounced 3 h after the injection. The effect of 200 mg/kg of the extract on carrageenan-induced paw edema showed greater anti-edema effect relative to the reference drug (indomethacin). The ethanolic extract also appeared to have a higher anti-edema effect than its aqueous counterpart (Table 4). The effect of the aqueous extract (50 and 200 mg) on histamine-induced paw edema was more pronounced 2 h after histamine injection, whereas that of the extract (100 mg) and indomethacin (10 mg/kg) was more pronounced 3 h after the injection. In the case of the ethanolic extract, the three doses and the reference drug (indomethacin, 10 mg/kg) produced the most pronounced effect 3 h after carrageenan injection. The effect of 200 mg/kg of the extract on histamine-induced paw edema showed greater anti-oedema effect relative to indomethacin (Table 5). The aqueous extract caused a decrease in the number of writhes at doses of 50, 100 and 200 mg/kg when compared to the control. The 200 mg/kg dose of the extract and indomethacin demonstrated a high antinociceptive power at 72.8% and 88.4%, respectively. The three doses of the ethanolic extract also caused a decrease in the number of writhes when compared with the control. However, the 200 mg/kg dose of this extract showed antinociceptive power of 52.3%, whereas that of indomethacin was 85.8%. The aqueous extract seemed to have a higher antinociceptive effect when compared with the ethanolic extract (Table 6). Table 7 shows the effect of the aqueous extract of AP and indomethacin on the paw lick test on mouse induced by formalin. The extract exhibited dose-dependent inhibition of paw lick by mice especially in the early phase. However, the effect of indomethacin was higher than that
Table 3 Mineral contents (mg/100 g) of the plant A. paniculata. Na
K
Mg
Ca
Zn
Fe
Cu
Co
Cr
Mn
Pb
Cd
Ni
59.45
58.2
37.7
149.5
6.05
4.35
2.25
1.05
0.55
1.6
0.05
Nil
0.55
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Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata 5
Table 4 Anti-inflammatory activity of the aqueous and ethanolic leaf extracts of A. paniculata and indomethacin on histamine-induced oedema in the right hind paw of rats. Treatment
Aqueous extract Ethanolic extract
Time, h 0 1 2 3 0 1 2 3
Extract dose, mg/kg; indomethacin, 10 mg/kg 50
100
200
Indomethacin
23.4 ± 1.5 26.4 ± 1.3 (–0.8) 25.6 ± 0.9 (8.7) 24.8 ± 1.1(6.70) 22.6 ± 0.5 26.0 ± 0.7 (2.3) 25.8 ± 0.8 (4.4) 24.4 ± 0.9 (7.6)
21.6 ± 0.5 24.0 ± 0.7 (8.4) 23.4 ± 1.1 (7.3) 22.6 ± 0.9 (15) 21.8 ± 0.4 25.2 ± 0.4 (5.3) 24.2 ± 0.8 (10.4) 23.0 ± 0.7 (12.9)
23.0 ± 1.2 24.2 ± 1.1 (7.6) 23.4 ± 0.9 (15.2) 23.4 ± 1.5 (12) 22.0 ± 1.7 24.2 ± 1.6 (9.0) 23.8 ± 1.9 (11.9) 22.2 ± 1.6 (15.9)
22.4 ± 0.5 25.4 ± 0.9 (3) 25.2 ± 0.8 (8.7) 24.2 ± 1.3 (9) 22.2 ± 0.4 25.2 ± 0.8 (5.3) 24.2 ± 1.3 (10.4) 23.6 ± 1.5 (10.6)
Control (2 mL/kg) 23.2 ± 1.1 26.2 ± 0.8 27.6 ± 1.5 26.6 ± 0.9 22.6 ± 0.9 26.6 ± 1.3 27 ± 1.4 26.4 ± 1.5
(n = 4), mean ± SD. Table 5 Anti-inflammatory activity of the aqueous and ethanolic leaf extracts of A. paniculata and indomethacin on carrageenan-induced edema in the right hind paw of rats. Treatment
Aqueous extract Ethanolic extract
Time, h 0 1 2 3 0 1 2 3
Extract dose, mg/kg; indomethacin, 10 mg/kg 50
100
200
24.4 ± 0.5 28.2 ± 0.8 (0.7) 27.2 ± 0.4 (2.1) 25.0 ± 1.0 (8.8) 24.4 ± 0.5 28.2 ± 0.8 (–0.7) 27.2 ± 0.4 (2.8) 25.0 ± 1.0 (11.4)
22.6 ± 1.8 25.6 ± 1.3 (9.9) 24.0 ± 1.4 (2.1) 22.8 ± 1.1 (16.8) 22.6 ± 1.8 25.6 ± 1.3 (8.6) 24.0 ± 1.4 (14.3) 22.8 ± 1.1 (19.2)
22.4 ± 1.1 23.0 ± 1.7 (19) 22.6 ± 1.1 (18.7) 22.4 ± 1.1 (18.3) 22.4 ± 1.1 23.0 ± 1.6 (17.9) 22.6 ± 1.1 (19.3) 22.4 ± 1.1 (20.6)
Control (2 mL/kg)
22.4 ± 0.9 28.4 ± 0.7 27.8 ± 1.6 27.4 ± 1.7 22.4 ± 0.5 28.0 ± 1.0 28.0 ± 1.4 28.2 ± 1.6
Indomethacin
22.8 ± 0.8 25.2 ± 1.5 (10) 24.0 ± 1.2 (14) 23.6 ± 1.1 (14) 23.0 ± 0.7 24.6 ± 0.5 (12.1) 23.8 ± 0.8 (15) 23.4 ± 1.1 (17)
(n = 4), mean ± SD.
of the highest dose of the extract. In contrast, the opposite was true between the reference drug and the 200 mg/kg dose of the extract in the late phase. The effect of the ethanolic extract on the paw lick test on mouse was comparable to that of the aqueous extract, except that the aqueous extract had a slightly higher antinociceptive activity than the ethanolic extract. Also in the late phase, the 200 mg/kg dose of the ethanolic extract showed a more antinociceptive effect than indomethacin (Table 8). In the case of the antioxidant activities of both extracts, the ethanolic extract showed a higher effect compared with the aqueous extract (Table 9).
Discussion The result of the acute toxicity experiments showed the absence of lethality or toxic side effects after oral administration of both aqueous and ethanolic extracts of A. paniculata even at the high dose of 1600 mg/kg. This result might be an indication of the non-toxic nature of the plant. In the sub-acute toxicity experiments performed on the aqueous crude extract of this plant in rats, it was found that the aqueous crude extract did not cause any significant changes in the red blood cell parameters and indices, although it caused significant changes in
Table 6 Analgesic effect of aqueous and ethanolic leaf extracts of A. paniculata and indomethacin on mouse writhing reflex induced by acetic acid. Treatment
Aqueous extract Ethanolic extract
50
100
16.0 ± 5.8 (68.9) 33.0 ± 6.2 (33.0)
16.0 ± 7.2 (68.9) 29.8 ± 6.2 (39.6)
Extract dose, mg/kg; indomethacin, 10 mg/kg 200 Indomethacin 14.0 ± 1.8 (72.8) 23.5 ± 3.7 (52.3)
(n = 4), mean ± SD. Percentage inhibition of writhes in parentheses.
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6.0 ± 4.1 (88.4) 7.0 ± 3.2 (85.8)
Control (2 mL/kg) 51.5 ± 14.1 (0) 49.3 ± 13.3 (0)
6 Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata Table 7 Analgesic effect of aqueous leaf extract of A. paniculata and indomethacin on paw lick test on mouse induced by formalin. Parameters
Control
52.5 ± 2.5 19.5 ± 0.6
Early phase Late phase
Indomethacin
9.8 ± 3.0 (81.3) 14.0 ± 8.5 (28.2)
Extract 50 mg
21.8 ± 1.7 (58.5) 17.0 ± 0.8 (12.8)
100 mg
20.3 ± 6.5 (61.3) 14.8 ± 3.8 (24.1)
200 mg
14.8 ± 4.1 (71.8) 6.3 ± 2.6 (67.7)
The extract (50, 100 and 200 mg/kg) and indomethacin exhibited higher inhibition at the early phase than at the late phase.
Table 8 Analgesic effect of ethanolic leaf extracts of A. paniculata and indomethacin on paw lick test on mouse induced by formalin. Parameters
Control
51.0 ± 1.8 19.8 ± 1.0
Early phase Late phase
Indomethacin
Extract
9.8 ± 3.0 (80.8) 14.0 ± 8.5 (29.3)
50 mg
100 mg
200 mg
25.0 ± 2.2 (51.0) 19.5 ± 1.7 (1.5)
29.3 ± 1.0 (42.6) 14.5 ± 5.4 (26.8)
14.0 ± 2.4 (72.6) 7.8 ± 3.8 (44.3)
(n = 4), mean ± SD. The extract (50, 100 and 200 mg/kg) and indomethacin exhibited high inhibition at the early phase.
white blood cell count and its differentials. The extract also caused significant changes in the levels of alkaline phosphatase, aspartate amino transferase, gamma-glutamyl transferase and alanine amino transferase. Increases were also observed in the levels of total protein, globulin, total bilirubin and unconjugated bilirubin. Histologically, the liver of the animals in some groups showed moderate vacuolar degeneration and necrosis of hepatocytes, whereas the kidney also showed eosinophilic casts in the tubules [29]. Thus, caution should be exercised in using this plant for medicinal purpose especially for prolonged periods. Inflammation induced by carrageenan is an acute one and it involves the synthesis or release of mediators, such as prostaglandins, serotonin, histamine, etc., at the injured site, and these mediators all cause pain and fever [30]. Anti-inflammatory drugs act by inhibiting these mediators from getting to the injured site, thereby ameliorating pain and inflammation. The highest antiinflammatory effect on carrageenan-induced edema was demonstrated by the 200 mg/kg dose of ethanolic extract 3 h after carrageenan injection. The extracts reduced the
oedema produced by histamine, suggesting that the antiinflammatory activity of the extracts is probably due to its anti-histamine activity. Histamine is a potent vasodilator that increases vascular permeability [31]. Therefore, the extracts could have acted either by inhibiting the synthesis of these inflammatory mediators or even their release from the storage sites. The results showed that all the extracts used in this study were effective. The anti-inflammatory and analgesic effects are comparable to that of indomethacin, the standard anti-inflammatory drug used. Phytochemical analysis of A. paniculata shows the presence of reducing sugars, saponin, phenolic group and alkaloids. The various phytochemical compounds detected are known to have beneficial importance in medicinal science. Phenols are said to offer resistance to diseases and accounts for most of the anti-oxidant activity in plants [32]. Flavonoids show anti-allergic, anti-inflammatory, anti-microbial and anti-cancer activities [33]. Alkaloids have been used to treat diseases such as malaria, and glycosides serve as defence mechanisms against many microorganisms. Saponin protects
Table 9 DPPH and ABTS radical scavenging activities, as well as FRAP and TP contents, of both aqueous and ethanolic leaf extract of A. paniculata.
DPPH (AAE)
FRAP (AAE)
TP (GAE)
ABTS (GAE)
Aqueous extract Ethanol extract
9.28 ± 0.002 (11.96%) 12.91 ± 0.007a (76.6%)
5.00 ± 0.47 (9.8%) 50.37 ± 0.23a (42%)
42.95 ± 0.02 (6.1%) 78.56 ± 0.04a (58.4%)
1.35 ± 0.07 (30%) 6.21 ± 0.003a (82.7%)
p < 0.001.
a
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Adedapo et al.: Biological activities of aqueous and ethanolic leaf extracts of A. paniculata 7
the plant against microbes and fungi. The plant is also known to contain carbohydrate, protein, crude fat, ash and crude fibre. The crude fibre content of the plant is high, and adequate intake of dietary fibre can lower the serum cholesterol level, risk of coronary heart disease, hypertension, constipation, diabetes, colon and breast cancer [34]. The relatively high level of carbohydrate, protein and others in this plant is attributed to its nutrient values. Analysis of the mineral content of this plant showed that it is rich in sodium, potassium, magnesium, calcium, zinc, iron, copper, cobalt, chromium, manganese, lead and nickel. Iron is an essential trace element for haemoglobin formation, normal functioning of the central nervous system and in the oxidation of fats, carbohydrates and protein. Therefore, consumption of the plant will increase the level of iron in the body. Although iron is an essential nutrient for plants, its accumulation within cells can be toxic. Thus, plants respond to both iron deficiency and iron excess by inducing expression of different gene sets [35]. Copper is naturally present in fruits and vegetables and is necessary for the proper growth and functioning of plants. The primary source of this mineral is from the soil, but small amounts may result from copper-based fungicides. Although copper concentrations in plants tend to increase with increasing copper concentrations in the soil, soil properties such as the acidity level and organic matter content can affect the amount of copper taken up [36]. Although the radical scavenging abilities of the extracts were less than those of ascorbic acid (85.3%) and gallic acid, the study showed that the extracts have proton-donating ability and could serve as free radical inhibitors or scavengers, acting possibly as a primary antioxidant. The ethanol extract particularly showed great promise with respect to free radical scavenging activity. Various phytochemical compounds detected from the plant are known to have beneficial importance in medicinal science [37]. Hence, the observed antioxidant activities may be due to the presence of these phytochemical compounds and nutritive values. Thus, the findings support the reported therapeutic use of this plant in traditional medicine for the alleviation of pain and inflammation [38]. Therefore, it could be concluded that both the aqueous and ethanolic extracts of A. paniculata have great medicinal potential, and this study may have justified the folkloric use of this plant for this purpose. Acknowledgments: This study was conducted with a research grant (SRG/FVM/2010A) from the University of Ibadan given to Dr. Adeolu Adedapo.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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