In vivo and in vitro pharmacological activity of Aristolochia tagala (syn: Aristolochia acuminata) root extracts

Abstract

Context: Aristolochia tagala Cham. (syn: Aristolochia acuminata Lam.) (Aristolochiaceae), known as Nallayishwari in Telugu, has been of interest to researchers because of its traditional uses for treating rheumatic pains and fever.

Objective: The anti-inflammatory activity of the petroleum ether, ethyl acetate, and ethanol extracts of A. tagala roots were investigated for the first time.

Materials and Methods: In vivo and in vitro anti-inflammatory effects were investigated employing the carrageenan-induced hind paw edema in rats and the macrophage cell line RAW264.7 stimulated with proinflammatory stimuli (lipopolysaccharide interferon γ or the calcium ionophore A23187) to determine PGE₂ or LTB₄ release, respectively.

Results: All the extracts exhibited anti-inflammatory effects which were found to be significant (p < 0.001) at 200 and 400 mg/kg, p.o, in rats tested and the ethyl acetate extract inhibited the induction of PGE₂ with IC₅₀ = 39.1 mg mL⁻¹ and LTB₄ with IC₅₀ = 29.5 mg mL⁻¹.

Discussion and conclusion: These findings demonstrate that the A. tagala roots have excellent anti-inflammatory activity and validate the traditional indications of this plant in its origin country.

Keywords::

• Anti-inflammatory activity
• kaempferol
• PGE₂
• LTB₄

Introduction

Aristolochia tagala Cham. (syn: Aristolochia acuminata Lam.) (Aristolochiaceae) is commonly known as “Native Dutchman’s Pipe” and in Telugu it is called as “Nallayishwari” (Ambasta, 2000). It is widely distributed in tropics and warm temperate zones, excluding Australia. The Aristolochiaceae has 7 genera and about 500 species. It is a tall climber shrub which grows up to 10–20 m height (Evans, 2009). Traditionally, the root portion is given for gastrointestinal complaints such as dyspepsia, flatulence, stomachaches, and snakebites (Bakshi et al., 1999), for treating fevers (including malarial fever), diarrhea (Prajapati et al., 2006), inflammation, and rheumatism (Kumar et al., 2002). It is also used as galactagogue (Pullaiah, 2002) and emmenagogue (Padua et al., 1999).

So far, phytochemical investigation was not carried out on this plant. As there is no scientific evidence in support of these claims, the root extracts of this plant and an active constituent, kaempferol, isolated from ethyl acetate extract, were subjected to in vivo and in vitro anti-inflammatory activity.

Materials and methods

Plant material

Plant material was collected from hilly regions of Bandarugudem Village, Krishna District, AP, during August to November 2008 and authenticated by Prof. M. Venkaiah, Department of Botany, College of Science and Technology, Andhra University, Vishakapatnam, AP, India. Voucher specimens were deposited in the herbarium of University College of Pharmaceutical Sciences (UCPS 865 dated 30 November 2008), Andhra University.

Preparation of extract

Shade-dried and powdered root (1.5 kg) was extracted with petroleum ether, ethyl acetate, and ethanol at temperatures of 40–60, 75–80, and 78–85°C, respectively, and the extracts were concentrated to obtain a semisolid residue (yield: 30, 45, and 56, respectively). Phytochemical screening (Harborne, 2009) gave a positive test for triterpenes and flavonoids.

Isolation of AT-1

The ethyl acetate extract was further subjected to column chromatography. Silica gel (100–200 mesh) (400 g) was packed in a column with pure hexane. Then, 25 g of ethyl acetate extract of A. tagala was added. The elution was done with petroleum ether, petroleum ether-ethyl acetate (5, 10, 15, 25, 50, and 75%) and ethyl acetate to give a total of 700 fractions (100 mL each). Twenty-five fractions (from 581 to 606) showed a single spot when they were subjected to thin-layer chromatography. All the 25 fractions combined and recrystallized which resulted in the isolation of AT1.

Reagents

Carrageenan, indomethacin, dimethyl sulfoxide (DMSO), interferon γ (IFNγ), bacterial lipopolysaccharide (LPS), calcium ionophore A23187, nordihydroguaiaretic acid (NDGA), and all other reagents were obtained from Sigma (St. Louis, MO).

Animals

Swiss albino mice (20–25 g) and Wistar albino rats (200–250 g) of either sex maintained in the Animal Experimental Laboratory of University College of Pharmaceutical Sciences, Andhra University, Vishakapatnam, AP, India under standard animal housing conditions (temperature 25 ± 2°C, relative humidity 75 ± 5%, and 12 h light and dark cycle) were used for experiments. The animals had access to standard laboratory feed and water. The study was approved by the Institutional Animal Ethical Committee, Ministry of Culture, Government of India (516/01/a/CPCSEA).

Acute toxicity

Mice were divided into five groups, each containing four animals. A. tagala extracts were administered orally at doses ranging from 0.5 to 8.0 g/kg following a standard method (Turner, 1965). Animals were continuously observed for 48 h to detect changes in the autonomic or behavioral responses and then monitored for any mortality for the following 7 days. A group of animals treated with the vehicle (0.5% carboxy methyl cellulose sodium) served as control. Based on the results of preliminary toxicity testing, the doses of 100, 200, and 400 mg/kg were chosen for further experiments.

Anti-inflammatory study

Carrageenan-induced rat paw edema

According to the method (Winter et al., 1962), the animals were fasted 18 h before the experiment and divided into different groups. Group A served as vehicle control given with 0.5% sodium carboxymethyl cellulose and group B served as positive control received indomethacin 50 mg/kg. Groups C, D, and E received A. tagala petroleum ether extract 100, 200, and 400 mg/kg, groups F, G, and H received A. tagala ethyl acetate extract 100, 200, and 400 mg/kg, and groups I, J, and K received A. tagala ethanol extract 100, 200, and 400 mg/kg. The paw volume of the rats was measured plesthysmographically before administering carrageenan (V₀) and 1, 2, 3, 4, 5, and 6 h after (V_t). The amount of paw swelling was determined for each rat and the difference between V_t (1, 2, 3, 4, 5, and 6 h) and V₀ was taken as the edema value. The percentages of inhibition were calculated according to the following formula (Garcia et al., 1995).

Cell viability

Cell viability was evaluated by using a 3-(4, 5-dimethyl-thiazole-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT)–based colorimetric assay (Diaz-Lanza et al., 2001). cells (8 × 10⁵) diluted with Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS) were pipetted into 96-well microtiter plates, and were incubated overnight at 37°C and 5% CO₂. They were exposed to various concentrations of samples for 3 h under the same conditions of incubation. MTT solution dissolved in phosphate-buffered saline was added to the wells at 1 mg mL⁻¹ final concentration. After carefully aspirating the medium, 100 µL of DMSO was added for the dissolution of formazan crystals. The absorbances of all wells were then read at 520 nm using a microplate reader. The values of the maximum non-toxic concentration obtained were used in PGE₂ and LTB₄ determination.

Assay of PGE₂ and LTB₄ release in A23187-stimulated macrophages

RAW264.7 cells were suspended in DMEM supplemented with 10% FCS, and pipetted into 24-well plates at a concentration of 5 × 10⁵ cells mL⁻¹. After adhering to plates (24 h at 37°C in an atmosphere of 5% CO₂), non-adherent cells were washed off, and the cells (90% of adherent cells) received fresh DMEM (without FCS). Cells were pre-treated for 1 h at 37°C with the test compounds or vehicle, and then stimulated further for 4 h by adding calcium ionophore A23187 (final concentration 10⁻⁶ M, for LTB₄ release) or 24 h with LPS IFNγ (100 ng mL⁻¹ and 10 U mL⁻¹, for PGE₂ release), respectively, according to Hulkower et al. (1996). Controls contained only DMSO (basic level of released eicosanoid) and reference compound (total inhibition of eicosanoid release). The contents of all dishes were frozen at −20°C and retained for analysis of PGE₂ and LTB₄ by means of a radioimmunoassay as stated in the procedure described by the manufacturer (Amersham, Buckinghamshire and UK).

Statistical analysis

The data are expressed as mean ± SEM. Student’s t-test was used for statistical evaluation.

Results

Characterization of AT-1

¹H-NMR and ¹³C-NMR experiments were performed to the compound AT-1. It is a yellow solid and its m.p. is 279–280°C. ¹H-NMR spectrum (400 MHz, acetone-d₆, δ, ppm, J/Hz): 8.05 (2H, d, J = 8.9, H-2′, 6′), 6.81 (2H, d, J = 8.9, H-3′, 5′), 6.08 (1H, d, J = 2.1, H-8), 6.29 (1H, d, J = 2.1, H-6). ¹³C-NMR spectrum (400 MHz, acetone-d₆, δ, ppm, J/Hz): 146.7 (C-2), 136.1 (C-3), 176.2 (C-4), 159.9 (C-5), 97.9 (C-6), 164.3 (C-7), 93.6 (C-8), 156.5 (C-9), 103.3 (C-10), 121.9 (C-1′), 129.3 (C-2′), 115.5 (C-3′), 159.4 (C-4′), 115.5 (C-5′), 129.3 (C-6′).

The ¹H-NMR spectrum of AT-1 revealed two sets of singlet’s at δ 6.08 (1H, d, J = 2.1 Hz) and 6.29 (1H, d, J = 2.1 Hz), these peaks have been assigned to H-6 and H-8, respectively. The presence of a set of doublets at 8.05 (2H, d, J = 8.9 Hz) and 6.81 (2H, d, J = 8.9 Hz) each integrating for two protons have been assigned to H-2′, 6′ and H-3′, 5′, respectively. ¹³C-NMR spectrum of AT-1 showed six oxygenated carbon atoms at δ 146.7, 136.1, 176.2, 159.9, 164.3, 156.5, and 159.4 assignable to C-2, C-3, C-4, C-5, C-7, C-9, and C-4′. The signal at δ 176.2, assigned to C-4, suggested the presence of flavonoid type of skeleton for AT-1. Thus, the above signals helped in assigning the structure to the compound AT-1 as kaempferol.

Acute inflammation carrageenan-induced paw edema assay

In carrageenan-induced paw edema method, the standard anti-inflammatory agent indomethacin (50 mg/kg) and the ethyl acetate and ethanol extracts at doses 200 and 400 mg/kg from roots of A. tagala produced a significant reduction in the volume of paw edema. But the petroleum ether extract did not produce any significant reduction (Table 1 and Figure 1).

Table 1.  Effect of drug vehicle, indomethacin, ATPE 100, 200, and 400 mg/kg, ATEAE 100, 200, and 400 mg/kg, and ATEE 100, 200, and 400 mg/kg on carrageenan-induced paw edema in rats of groups A–K, respectively.

Treatment groups	Percentage inhibition of the maximal paw edema during 6 h	Percentage inhibition of total AUC paw edema during 6 h
A	0.0 ± 4.19	0.0 ± 1.10
B	68.52 ± 1.86***	84.05 ± 1.35***
C	31.23 ± 2.42*	40.54 ± 1.65*
D	34.13 ± 1.31*	49.22 ± 1.40*
E	48.23 ± 7.17**	65.46 ± 0.53***
F	50.32 ± 3.2***	68.54 ± 1.55***
G	55.27 ± 1.58***	78.10 ± 2.38***
H	64.16 ± 2.92***	80.01 ± 1.22***
I	42.76 ± 1.34**	50.23 ± 2.45**
J	47.90 ± 3.92**	62.96 ± 0.75***
K	59.78 ± 1.06***	74.41 ± 0.41***

Values are mean ± SEM, n = 4; *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; Student’s t-test.

Figure 1.  Effect of the petroleum ether, ethyl acetate, and ethanol extracts of A. tagala at doses of 400 mg/kg along with indomethacin (50 mg/kg body wt) on the total paw edema in carrageenan-induced rats. Values are mean ± SEM, n = 4; p < 0.05, p < 0.01, p < 0.001 vs. control; Student’s t-test.

Effects of PGE₂ and LTB₄ release

A. tagala extract, indomethacin and NDGA were not cytotoxic at any of the concentrations tested (1–100 mg mL⁻¹). Addition of LPS IFNγ to RAW264.7 macrophages causes the generation of nanogram amounts of eicosanoids via both cyclooxygenase (COX) and lypoxygenase (LOX) pathways as shown in Table 2. This effect was measured in terms of immune assayable PGE₂ and LTB₄, respectively. Validation of this system for the identification of inhibitors of the two divergent pathways of arachidonate metabolism was performed by using indomethacin, a well-characterized COX inhibitor (93.2% inhibition of PGE₂ at 100 µM) and NDGA, a known inhibitor of 5-LOX (94% inhibition of LTB₄ at 25 µM). The compounds tested showed a considerable activity as inhibitors of eicosanoid release from RAW264.7 macrophages stimulated by LPS IFNγ and calcium ionophore A23187. The IC₅₀ values for A. tagala were 39.1 mg mL⁻¹ (PGE₂-release assay) and 29.5 mg mL⁻¹ (LTB₄-release assay).

Table 2.  Effect of Aristolochia tagala ethyl acetate (EA) extract, kaempferol, and indomethacin on PGE₂ and LTB₄ biosynthesis by RAW264.7 macrophages stimulated by LPS IFNγ and A23187.

	Concentration	PGE2 (ng mL^−1)	Inhibition (%)	LTB4 (ng mL^−1)	Inhibition (%)
LPS + IFN	100 ng mL^−1+ 10 U mL^−1	208.0 ± 12.5	0	—	—
A23187	10^−6 M	—	—	47.8 ± 8.8	0
A. tagala EA extract	10 mg mL^−1	195.0 ± 13.2	6.2	33.8 ± 8.4	29.2
	25 mg mL^−1	148.5 ± 9.8*	28.6	27.2 ± 5.7*	43.1
	50 mg mL^−1	70.6 ± 2.6*	66.0	9.0 ± 3.4*	81.1
	100 mg mL^−1	25.5 ± 3.2*	87.7	4.1 ± 2.9*	91.4
Kaempferol	5 mg mL^−1	45.2 ± 2.1*	78.27	8.1 ± 3.2*	83.06
	10 mg mL^−1	20.3 ± 1.9*	90.25	3.5 ± 1.5*	92.68
Indomethacin	100 μM	14.1 ± 3.2*	93.2	—	—
NDGC	25 μM	—	—	2.9 ± 0.4	94.0

RAW264.7 cells were pre-treated for 1 h at 37°C with A. tagala EA extract, indomethacin, nordihydroguaiaretic acid and vehicle (dimethyl sulfoxide). Then, they were stimulated for a further 4 h by calcium ionophore A23187 (10⁻⁶ M) for LTB₄ release or 24 h by LPS (100 ng mL⁻¹) IFNγ (10 U mL⁻¹) for PGE₂ release, according to the procedure described in Materials and Methods section. LTB₄ and PGE₂ were determined by means of radioimmunoassay. Values represent mean ± SEM (n =  4).

*p < 0.01 compared with control groups.

Discussion

Among several traditional claims, the effectiveness of Nallayishwari in fever and inflammation has been particularly emphasized (Padua et al., 1999). Hence, it was considered that pharmacological investigation on A. tagala for these properties was deserved to scientifically validate the traditional claims.

The phytochemical profile carried out in this study pointed out the presence of triterpenes and flavonoids. The results of acute toxicity testing indicate that all the extracts of A. tagala are safe even at an oral dose of 8 g/kg.

In vivo studies revealed that all the extracts of A. tagala exhibit anti-inflammatory properties, as there was a significant inhibition of the acute edema in carrageenan-induced paw edema rats. Carrageenan-induced paw edema is a test mostly used to study anti-inflammatory drugs, both steroidal and non-steroidal, as it involves several mediators. This kind of test brings about an inflammatory reaction in two different phases. The initial phase, which occurs between 0 and 2.5 h after injection of the phlogistic agent, has been regarded as belonging to the action of histamine, serotonin, and bradykinin on vascular permeability (Vinegar et al., 1987). The edema volume reaches its maximum approximately 3 h post-treatment and then begins to decline. The late phase, which is also a complement-dependent reaction, has been shown to be a result of over production of prostaglandins in tissues (Di Rosa, 1974). As the ethyl acetate extract shows a higher inhibition of maximal paw edema than the other two extracts, it was subjected to column chromatography for separation of pure constituent. By examining the spectral data of the pure constituent isolated from the ethyl acetate extract, it was named as kaempferol (Figures 2 and 3). The A. tagala extracts, inhibited the edema from the first hour, acting in both the earlier phase and the later one.

Figure 2.  ¹H-NMR spectrum of kaempferol.

Figure 3.  ¹³C-NMR spectrum of kaempferol.

In vitro studies were carried out on eicosanoid-releasing system to investigate whether the extracts and kaempferol contribute to this mechanism of antiinflammatory activity. Many different studies have revealed that macrophages are a potent source of arachidonic acid metabolites generated via COX and LOX pathways (Wei & Frenkel, 1992). Besides, macrophages participate in host defense, immunity, and inflammatory responses where they are potently activated resulting in the production of cytokines, oxygen, and eicosanoids. In macrophages, LPS alone or in combination with cytokine like IFNγ and calcium ionophore A23187 are the best characterized stimuli to bring about proinflammatory proteins, resulting in cytokine release and synthesis of enzymes such as COX-2 and 5-LOX, respectively (Garrido et al., 2004). The inducible isoform such as COX-2 and 5-LOX would be responsible for the high prostanoid generation during inflammatory responses (Herencia, 1999). Kaempferol has inhibited in vitro PGE₂ and LTB₄ release from calcium ionophore and LPS IFNγ–stimulated macrophages higher than all the doses of ethyl acetate extract and standard drug (Table 2). Its percentage inhibitions were 87.7% (PGE₂ release) and 91.4% (LTB₄ release) at a dose of 10 mg mL⁻¹, respectively, and indomethacin only exhibited inhibitory activity on PGE₂ but not on LTB₄. With these in vivo and in vitro studies, we could say that the ethyl acetate extract as well as its pure constituent, kaempferol, possess inhibitory action on both COX and LOX enzymes. This indicates that all the doses of the extracts could inhibit different aspects and chemical mediators of inflammation (histamine, serotonin, bradykinin, prostaglandins). The results were significant when analyzed statistically. Thus, the extracts showed anti-inflammatory activity at various phases of inflammation.

Conclusion

Our work suggests that the petroleum ether, ethyl acetate, and ethanol extracts from roots of A. tagala possess an anti-inflammatory effect that could be similar to the COX and lipoxygenase inhibitors. The active constituent, kaempferol, is responsible for anti-inflammatory activity of ethyl acetate extract as it was inhibited by PGE₂ and LTB₄. These data proved the validity of the traditional indications of this plant in its origin country. Therefore, the extracts of A. tagala will be of great potential benefit in the management of inflammatory disorders.

Acknowledgements

We are very grateful to Dr. M. Venkaiah, Professor, Department of Botany, College of Science and Technology, Andhra University, Vishakapatnam, AP, India for his valuable information on identification of plant material.

Declaration of interest

The authors report no declaration of the interest.