Leukocyte-Stimulating Effect and Phytochemical Screening of Trichilia hirta Extracts

T richlia hirta L. (Family Meliaceae) is a tree largely distributed in South America and the Caribbean. Their leaves and roots have been used in traditional medicine to treat tumors and external ulcers. ^1,2 Extracts from T. hirta have been also been reported being antimalarial and anti-inflammatory. ^3,4 Phytochemical research on T. hirta has shown the presence of limonoids, protolimonoids, polyphenols, and saponins. ^5,6 Extracts from this plant are extensively used as an antitumor resource for patients with cancer in Santiago de Cuba, Cuba. ⁶ However, knowledge is limited on the phytochemical composition of the plant organs commonly used and the properties of the extracts on the immune system.

Immunomodulation is a procedure that can alter the immune system of an organism by interfering with its functions of protection; if it results in an enhancement of immune reactions, the compound is termed an immunostimulative drug that primarily implies stimulation of nonspecific systems. ⁷

The present study evaluated the leukocyte-stimulating effect of T. hirta root extracts on BALB/c mice. The study also determined the phytochemical composition of the ethanol extracts obtained from leaves, roots, and stem bark of this species.

Leaves, roots, and stem bark of T. hirta were collected in Santiago de Cuba, Cuba, and identified by a specialist at the Eastern Center for Ecosystem and Biodiversity. A voucher specimen (BIOECO number 1078) was deposited at the herbarium of this institution.

Air-dried plant organs, stem bark, roots, and leaves (50 g), respectively, were soaked in 250 mL of 60% ethanol for 7 days and filtered, to obtain the ethanol extract of each organ of T. hirta. Another ethanol extract from roots (50 g) was prepared and concentrated under reduced pressure by rotary evaporation (RV Basic model, IKA, Staufen, Germany).

The ethanol extracts of T. hirta were subjected to preliminary phytochemical screening for detection of several plant phytoconstituents. ⁸

Total phenolic content was obtained by the Folin–Ciocalteu method, and the absorbance was read at 765 nm using tannic acid as a standard. ⁹ The phenol content was expressed as equivalents of tannic acid (in mg/g) per gram of dry extract. The total carbohydrate content of the extracts was determined by the phenol-sulfuric acid method, and the absorbance was read at 492 nm using glucose as a standard. ¹⁰ The carbohydrate content was expressed as mg equivalents of glucose per gram of dry extract. Three determinations of these constituents were made for each extract.

Thirty-six female BALB/c mice weighing 18–22 g were housed under standard environmental conditions (25°C) with 10-hour/14-hour light/dark cycles and fed ad libitum with a conventional pelleted diet and water.

The mice were divided in three groups (n=12): two experimental groups orally treated with ethanol extract at 82 and 976 mg/kg, respectively, from root for 7 consecutive days and a control group of animals without treatment. The ethanol extract from the root was selected for biological evaluation because cancer patients in Cuba frequently use this organ. ¹¹ All experiments were conducted according to a protocol approved by the Local Animal Care Committee.

Blood samples were collected by retro-orbital puncture on days 2, 4, and 7. The cell counts (total and differential leukocytes) were carried out according to the standard protocol with a Neubauer chamber. ¹²

All data were expressed as mean±SEM values. The significance of difference between means (P<.05) was measured by Student's t test.

Saponins and polyphenols were identified in leaves and root of T. hirta (Table 1); similar results have been previously reported. ¹³ Bioactive phytochemicals, saponins, and coumarins were detected at high concentrations in leaves and root (Table 1). Other compounds identified in the ethanol extracts were flavonoids, tannins, alkaloids, quinones, and glycosides. The yields of carbohydrate and phenol constituents increased drastically when the ethanol extract was concentrated (Table 2).

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Table 1.

Phytochemicals Detected in T. hirta Ethanol Extract by Phytochemical Screening

Constituents analyzed	Leaves	Root bark	Root central cylinder	Stem bark
Alkaloids	++	+	−	+
Sterol and triterpenes	+	++	+	+
Quinone	+	+	−	++
Coumarins	+	+++	−	++
Saponins	+++	++	−	−
Lipids	++	+	++	+
Reducing sugars	+++	++	+	+++
Phenols/tannins	+	+	−	+
Tannins	+	+	−	+
Amino acids	+++	+++	+	++
Flavonoids	+	+	−	+
Carbohydrates/glycosides	+	++	+	++

The ethanol extracts of T. hirta were subjected to preliminary phytochemical screening for detection of several plant phytoconstituents. Detection was rated on the following scale: not detected (–) or detected with a concentration of low (+), moderate (++), or high (+++).

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Table 2.

Carbohydrate and Phenol Contents of the T. hirta Extracts

		Content
Dried extract	Yield (%) a	Carbohydrate (μg/g)	Phenols (μg of TAE/g)
Ethanol extract	0.06	21.1±1.378	0.120±0.003
Ethanol extract concentrated	0.72	303.6±13.555	1.312±0.022

Total phenolic content was obtained with the Folin–Ciocalteu reagent and is expressed as equivalents of tannic acid (TAE) (in μg/g) per gram of original weight of the root. The total carbohydrate content of the extracts was determined by the phenol-sulfuric acid method and is expressed as mg of glucose per gram of original weight of the root of T. hirta. Data are mean±SEM values.

a

The percentage yield was calculated with reference to the original weight of the root of T. hirta.

Administration of extract at 976 mg/kg to BALB/c mice increased total leukocytes by 15–33% in peripheral blood of healthy animals at days 2, 4, and 7 compared with the control group (Fig. 1). However, the dose of 82 mg/kg extract only showed a significant stimulating effect (6.13±0.59) at day 4 compared with the control value (P<.05).

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FIG. 1.

T. hirta extract showed a stimulating effect on total leukocyte, lymphocyte, and neutrophil counts in healthy BALB/c mice. Two experimental groups were orally fed with ethanol extracts at 82 and 976 mg/kg, respectively, for 7 consecutive days and compared with a control group of untreated animals. Blood samples were collected on days 2, 4, and 7. The cell counts were carried out according to the standard protocol with a Neubauer chamber. *Significantly different from the control group (P<.05).

These stimulant effects could be related wto the adjuvant activity of some phytochemicals identified in T. hirta. In fact, saponins, flavonoids, and glycosides in general have been reported as immunostimulants. ^{14 –16}

The extract dosage of 82 mg/kg increased the neutrophil counts at days 4 and 7 compared with the controls (Fig. 1). The dosage of 976 mg/kg significantly increased the neutrophil counts at day 7 (P<.05). These findings suggest that T. hirta extracts could help to increase the immunity against microbial infections. ¹⁷

A decrease of lymphocyte counts compared with the control group was observed during the study. This fact could be related to the migration of lymphocytes to secondary lymphoid organs, like lymphatic ganglions and spleen, where these cells exert their immunological functions.

The effects on neutrophil and lymphocyte counts imply that the observed increase for total leukocytes was associated with the increase in the neutrophil population (Fig. 1). This suggests a nonspecific mechanism of immunostimulation where neutrophils are particularly affected by the stimulant effect of phytochemicals present in ethanol extracts.

In general, T. hirta extracts possess several phytochemical families reported as immunostimulant and antitumor. The oral administration of T. hirta extracts to BALB/c mice suggests that ethanol extracts have a leukocyte-stimulating effect. These results contribute to the validation of the traditional use of this plant.