Bioactive Properties of Tynanthus panurensis (Bureau) Sanwith Bark Extract,the Amazonian “Clavo Huasca”

Abstract

Tynanthus panurensis (Bureau) Sanwith (Bignoniaceae) is a liana vine used in traditional Amazonian medicine as a tonic and energizer as well as a treatment for rheumatism. These traditional indications prompted this study of the antioxidant and anti-inflammatory activities of T. panurensis bark extract (ETP). Phytochemical analysis of ETP showed the presence of saponins and a high concentration of phenols and flavonoids. A battery of in vitro tests revealed that the extract has free radical–scavenging antioxidant properties and reduces microsomal lipid peroxidation, uric acid synthesis, and tumor necrosis factor-α production. The anti-inflammatory properties of ETP were further confirmed in vivo in a rat carrageenan edema model, in which the extract exhibited a potent activity. These results support the idea that T. panurensis bark extract could be beneficial for treating inflammation and are in agreement with one of the main traditional uses of this plant.

Introduction

N atural products, especially plants, have been used to treat various diseases for thousands of years. Traditional herbs are still widely used, and they represent one of the main sources of bioactive products of therapeutic interest. Such areas of interest include capacity as an antioxidant for inflammation and other abnormalities associated with the generation of reactive oxygen species and for induction of lipid peroxidation.¹ Antioxidants play an important role in reducing oxidative stresses; they can help protect against these diseases by scavenging free radicals and reducing hydrogen peroxide.^2,3 Accordingly, there is increasing interest in these products, both to avoid the effects of free radicals in the human body and to prevent the deterioration of fats and other constituents of food.

Authors have expressed preference for antioxidants from natural sources, which might be safer than synthetic compounds⁴ and could then provide new medicines and food additives.⁵ Several studies have revealed that plants can produce safe and potent antioxidant compounds, especially phenolic compounds and flavonoids.⁶ Phenolic compounds are efficient scavengers of free radicals and blockers of lipid peroxidation; they can chelate transition metals, thereby preventing the redox cycle of these metals.^7,8 Given this context, we sought to determine whether the extract of Tynanthus panurensis could be a source of this type of product and thus possess anti-inflammatory activity. This hypothesis is based on the traditional use of the plant.

T. panurensis (Bureau) Sanwith (Bignoniaceae) is a large liana vine indigenous to the Amazon rainforest and other parts of tropical South America. The plant is common called “clavo huasca” and is still widely used as a natural aphrodisiac in Peruvian herbal medicine.⁹ The powder of the dried stem is used as a spice in the same way as cinnamon, and its bark tincture is indicated as a remedy for numerous diseases (such as arthritis, rheumatism, inflammation, and digestive problems), an appetite enhancer, and a digestive stimulant. Despite these uses, a survey of the literature revealed that experimental studies on the anti-inflammatory and antioxidant activities of this plant are still lacking. In this study we evaluated the antioxidant and scavenging activities of an extract of T. panurensis bark (ETP) by using 3 experimental assays: (1) bleaching of the stable 1,1Í-diphenil-2-picrylhydrazyl radical (DPPH); (2) scavenging of superoxide anion, generated by xanthine/xanthine oxidase system; and (3) microsomal lipid peroxidation in presence of iron and ascorbic acid. These experiments were followed by an in vivo study of the potential anti-inflammatory activity of the extract in the rat carrageenan test. In addition, an initial phytochemical analysis of ETP was performed to detect potentially bioactive phenols and flavonoids, which could extend the list of compounds of interest already known to be present in this plant (i.e., tinantina, eugenol, phenylpropanoid glycosides, and tannic acids).¹⁰

Materials and Methods

Plant material and preparation of the extract

The bark of T. panurensis was acquired from a traditional market in Iquitos, Peru, one of the most important sources of medicinal plants in Amazonia.¹¹ The plant material was identified and authenticated by the Botanical Department of Universidad CEU–San Pablo, and a voucher specimen was kept in our herbarium under reference number 250399. A crude extract was obtained by shaking the powdered bark (250.0 g) in methanol/water (4:1) for 30 minutes. After this, the extract (ETP) was collected and concentrated until dried under reduced pressure at 40–45°C. The final product (yield 13.75%, w/w) was then stored at 4°C.

Preliminary phytochemical analysis

A preliminary phytochemical analysis was done according to the methods of Pascual et al.¹² The separation was performed on silica gel plates (60 F254; Merck) by using 3 solvent systems, each applied for the identification of their polarity. Spots were visualized first under ultraviolet 254-nm and 365-nm light and later were sprayed with different specific reagents (Dragendorff reagent, potassium hydroxide, Liebermann–Burchard reagent, natural products (Diphenyl boryloxy ethylamine)/polyethylene glycol reagent, and iron [III] chloride reagent).

Determination of total phenolic content

The total phenolic content in the ETP samples was determined by using Folin–Cicolteau reagent.¹³ To 200 μL of the extract (1.3 mg/mL), 1.0 mL of 10% Folin–Cicolteau reagent and 800 μL of 7% Na₂CO₃ were added and mixed. After 2 hours, the absorbance was measured at 750 nm. A standard curve of gallic acid (1.0–6.25 μg/mL) was used for quantification. The concentration in the extract was expressed as micrograms of gallic acid equivalents.

Determination of total flavonoid content

The flavonoid content was determined according to the method of Zhishen et al.¹⁴ by using quercetin as standard (1.0–6.2 μg/mL). For an initial mixture (1.0 mL), 200 μL of the extract (1.3 mg/mL) and 60 μL of 5% NaNO₂ were added (time 0). At 5 minutes, 60 μL of 10% AlCl₃ was added; at 6 minutes, 400 μL of 1M NaOH was added. The absorbance of the mixtures was measured at 510 nm. The concentration of flavonoid compounds was expressed as quercetin equivalents.

DPPH-scavenging activity

Different concentrations of the extract, gallic acid, quercetin, and Trolox (Hoffman-LaRoche) were incubated in a 96-well microtiter plate, in darkness, with DPPH (1 mM in methanol). After 20 minutes of incubation at room temperature, the absorbance was measured at 517 nm in a microplate reader (Versa Max; Molecular Devices).¹⁵ The results were expressed as vitamin C equivalent antioxidant capacity (VEAC) in mg/100 mg of extract.¹⁶

Superoxide anion–scavenging activity

The scavenging potential for superoxide radicals was analyzed via a hypoxanthine/xanthine oxidase–generating system coupled with nitroblue tetrazolium (NBT) reduction as described McCune and Johns.¹⁷ Microplates with the reaction mixture were read at 540 nm by using a microplate reader (Versa Max) 2.5 minutes after the addition of xanthine oxidase (1 unit per 10 mL buffer) and every 5 minutes for 40 minutes. A negative control was prepared without plant extract. To ensure that there was no direct NBT reduction by the extract compounds, a control was made by mixing NBT solution with each extract in phosphate buffer. The possibility that NBT reduction was due to inhibition of xanthine oxidase rather than superoxide scavenger activity was also checked by spectrophotometrically¹⁸ monitoring the rate of uric acid formation in the absence and presence of extracts at 295 nm every 5 minutes for 40 minutes. Superoxide scavenging ability was expressed as percentage inhibition of NBT reduction in samples with the extract compared with control, as well as VEAC values.

Microsomal lipid peroxidation

Microsomes were isolated from homogenized livers of Wistar rats. Their peroxidation in the presence of iron and ascorbic acid was measured by the thiobarbituric acid method as described by De las Heras et al.¹⁹ The ETP concentration tested was 100 μg/mL, and butylated hydroxytoluene was used as the reference compound (100 μM).

Tumor necrosis factor-α production

Human promyelocytic leukemia cells (HL-60 cell line) were obtained from American Type Culture Collection (CCL-240) and maintained in our laboratory. Cell production of tumor necrosis factor (TNF)-α was measured by using an enzyme-linked immunosorbent assay spectrophotometric kit (Amersham Pharmacia Biotech Ltd.) according to the manufacturer instructions. The dried extract was dissolved in phosphate-buffered saline at 3 different concentrations (100, 50, and 25 μg/mL). Cultures of 10⁵ cells were incubated with chemical inductors of TNF-α synthesis: 50 nM okadaic acid or 20 nM 2-o-tetradecanoyl phorbol-13-acetate in the absence or presence of extracts. Phosphate-buffered saline and thalidomide were used as negative and positive controls, respectively. After 24 hours of incubation (37°C, 5% CO₂ atmosphere), cells were centrifuged and supernatant was used in enzyme-linked immunosorbent assay.²⁰ The amount of the TNF-α production with inductors was defined as 100%. Results of a previous assay confirmed that the extract had no cytotoxic effect on HL-60 cells (data not shown).

Carrageenan-induced edema of hind paw

Male Sprague–Dawley rats (weighing 200–250 g and bred at Universidad CEU–San Pablo) were used for the study. The animals were housed in cages with water and food available ad libitum, were kept in a controlled environment (temperature, 20–22° C; dark/light cycle, 12 hours/12 hours; humidity, 45%–55%), and were randomly assigned to the different experimental groups. The assay was performed in accordance with the Guide for the Care and Use of Laboratory Animals promulgated by the National Institutes of Health.

Anti-inflammatory activity was determined by using the method of Winter et al.,²¹ as modified by Alguacil et al.²² At the beginning of the study, the baseline paw volume was determined by submerging the right hind paw up to the tibiotarsal joint into a water cell of a plethysmometer (Digital Pletysmomet, L37500; Letica). The volume of displacement, which is equal to the paw volume, was then read on a digital display. Each determination was the average of 3 repeated measures. After baseline determination, the rats were injected intraperitoneally with ETP (1 mg/kg); saline (control group) or indomethacin (5 mg/kg; Sigma) was used as a reference compound. One hour afterward, the rats were subcutaneously injected with 0.1 mL of 1% lambda carrageenan (Sigma) into the surface of the right hind paw. The effect on paw volume was studied 1, 2, and 3 hours after injection.

Statistical analysis

Statistical analysis was performed with one-way analysis of variance, followed by multiple range tests (least-squares difference test, using STATGRAPHICS Plus software). Differences were considered statistically significant at a P value less than .05.

Results and Discussion

Many tropical plants have interesting biological activities with potential therapeutic applications. T. panurensis is largely used by local people in the Amazon rainforest as a tonic and energizer, as well to treat rheumatism. The bark of this liana vine has recently become more popular and can be found on the international market as dried power and an alcoholic preparation. Despite its long and popular use in South America, to our knowledge no published experimental or clinical studies support the therapeutical properties of the clavo huasca.

The phytochemical analysis of the ETP done by using thin-layer chromatography showed that the extract contained alkaloids, coumarins, antraquinones, and saponins. ETP was positive for phenolic compounds (mean±standard deviation, 226.76±1.99 mg gallic acid equivalents per g of extract) and flavonoids (mean±SD, 126.91±5.43 mg quercetin equivalents per g of extract). Therefore, ETP contents include potential antioxidants products that deserve further attention.

Several procedures can be used to evaluate the total charge of antioxidants present in a complex mixture of compounds, such as beverages, infusions, or plant extracts like ETP. In these cases, measuring the consumption rate of stable free radicals in comparison with a given reference compound (i.e., ascorbic acid) provides a suitable quantitative estimation of the total charge of antioxidants.²³ Several authors also suggest that more than one of these methods and reference compounds are needed to compare the antioxidant properties of different complex mixtures.^24,25 Accordingly, we evaluated the antioxidant activity of ETP by testing its ability to scavenge both DPPH and superoxide anions. In both cases we tested 3 reference compounds and the VEAC indexes. A significant scavenging activity of ETP was observed, even when the VEAC values were lower than those of the standard antioxidant (Table 1). In some cases this difference was small, and thus the VEAC value of ETP in the superoxide assay (0.025) was only moderately lower than that of Trolox (0.038).

Table 1.

DPPH Radical– and Superoxide Anion–Scavenging Activities of T. panurensis Extract, Quercetin, Gallic Acid, and Trolox

Sample	Tynanthus panurensis extract	Quercetin	Gallic acid	Trolox
DPPH	36.6±2.41	344.3±5.43	729.4±11.9	81.6±8.6
Superoxide anion	0.025±0.007	10.09±0.90	4.26±0.70	0.038±0.007

Values are ascorbic acid equivalents and are expressed as means±standard deviation of 3 measurements (P<.05).

DPPH, diphenil-2-picrylhydrazyl radical.

One of the main generation systems of superoxide radicals is the activation of xanthine-xanthine oxidase. In fact, several flavonoids and other phenolic compounds are considered antioxidants not only because they act as free radical scavengers but also because they inhibit xanthine oxidase.²⁶ That is why we examined the activity of ETP on xanthine oxidase activity in vitro and found that the extract effectively inhibited NTB reduction as well as uric acid formation (Table 2). The latter effect was more evident at 200 μg/mL, which provided a 65.67 % enzymatic inhibition. It seems, therefore, that ETP may act as both a scavenger and a xanthine oxidase inhibitor.

Table 2.

Effect of T. panurensis Extract on the Xanthine/Xanthine Oxidase System: Scavenging Activity and Xanthine Oxidase Inhibition

T. panurensis extract (μg/mL)	Inhibition of uric acid production (%) ^a	Inhibition of nitroblue tetrazolium reduction (%) ^a
200	40.76±2.51	65.67±2.51
100	35.03±2.01	49.25±1.56
50	28.80±1.74	25.87±1.23

Values expressed are means±standard deviation of 3 measurements (P<.05).

Apart from lowering free radicals, antioxidant substances can also act at other steps of the oxidative process. Thus, primary mechanisms consist of capturing free radicals or breaking molecular chains, whereas secondary or preventive mechanisms include metal deactivation, inhibition of lipidic peroxidation, or regeneration of primary antioxidant.²⁷ We further tested ETP (100 μg/mL) for its ability to inhibit nonenzymatic lipid peroxidation in rat liver microsomes stimulated by FeCl₃-ascorbate. The inhibitory effect of the extract (127.16%±14.18%) was prominent and even higher than that of the reference compound (butylated hydroxytoluene, 100 μM), thus suggesting that inhibition of lipid peroxidation could play a relevant role in the antioxidant action of ETP.

Much evidence has shown that reactive species, such as superoxide anion radical, hydrogen peroxide, hydroxyl radical, and peroxynitrite, are produced at sites of inflammation.²⁸ High concentrations of these reactive species generate an oxidative imbalance, decrease the capacity of the endogenous antioxidant enzymes to remove them and contribute to tissue damage in acute and chronic inflammation. Given the previously described antioxidant activity of ETP, an evaluation of its potential anti-inflammatory properties in vivo appeared highly recommendable. We chose the carrageenan-induced paw edema model for this purpose because it is perhaps the most widely used primary test for the screening of new anti-inflammatory agents.²¹ In this test, the early phase of inflammation (1–2 hours) is known to be mediated mainly by histamine, serotonin, and kinins and is followed by the release of prostaglandins; on the other hand, the second phase is more related to neutrophil infiltration and the production of the reactive free radical species.^29,30 In our experiments, preteatment with ETP significantly inhibited edema formation from the first hour until the end of the experiment (Table 3), a finding that confirms an anti-inflammatory effect of the extract and suggests that several mechanisms can simultaneously contribute to this action. Among these possible mechanisms we studied the effect of ETP on TNF-α production because this cytokine is a master regulator of inflammation and a key player in the cytokine network.³¹ In fact, ETP proved to be effective because it dose-dependently depressed TNF-α production in HL-60 cells stimulated with chemical promoters (Table 4).

Table 3.

Anti-Inflammatory Effect of T. Panurensis Extract on Carrageenan-Induced Hindpaw Inflammation in Rats

		Paw volume after carrageenan injection (mL) ^a
Treatment	Rats (n)	Baseline	1 Hour	2 Hour	3 Hour
Saline	10	1.69±0.1	2.23±0.2	3.14±0.3	3.43±0.4
Indomethacin	10	1.69±0.1	1.93±0.1^*	2.41±0.1^*	2.65±0.3^*
T. panurensis	9	1.72±0.1	1.74±0.1^*	1.72±0.1^**	1.72±0.1^**

Values are expressed as mean±standard deviation.

P<.05 vs. saline.

P<.05 vs. saline and indomethacin.

Table 4.

Effects of T. panurensis Extract on Tumor Necrosis Factor-α Production by Human Leukemia HL-60 Cells Induced with 50 nM Okadaic Acid or 20 nM 2-O-Tetradecanoyl Phorbol-13-Acetate

	Tumor necrosis factor-α inhibition (%) ^a
Exposure	Okadaic acid	O-tetradecanoyl phorbol-13-acetate
T. panurensis
100 μg/mL	34.08±4.66	36.43±5.66
50 μg/mL	32.12±3.75	33.72±7.24
25 μg/mL	14.18±2.67	22.91±2.36
Thalidomide	53.49±1.42	164.73±2.18

Amount of tumor necrosis factor-α inhibition with only o-tetradecanoyl phorbol-13-acetate or okadaic acid was defined as 100%. Values are expressed as means±standard deviation of 3 measurements (P<.05).

In summary, both in vitro and in vivo experiments show that T. panurensis bark extract posses antioxidant and anti-inflammatory properties involving different biological mechanisms. These results provide experimental evidence supporting the traditional use of this plant in various diseases associated with inflammation and suggest that it might be a source of new anti-inflammatory drugs.

Footnotes

Acknowledgments

We are grateful to the Universidad CEU San Pablo (grant no. 02/98 U.S.P.) for financial support and to Brian Crilly for his assistance in the preparation of this manuscript.

Author Disclosure Statement

No competing financial interests exist.

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