Isolation and Chemical Analysis of a Fatty Acid Fraction of Kalanchoe pinnata with a Potent Lymphocyte Suppressive Activity

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Ethanolic extraction
• Purification of EE
• KP12 purification
• Cells
• T Cell proliferation
• Results and Discussion
• Acknowledgements
• References

Abstract

Previously we demonstrated that Kalanchoe pinnata (KP) leaf extracts inhibited in vitro lymphocyte proliferation and showed in vivo immunosuppressive activity. Here we attempt to identify the immunosuppressive substances present in KP guided by the lymphoproliferative assays. From the ethanolic extract was purified a fraction (KP12SA) twenty-fold more potent to block murine lymphocyte proliferation than the crude extract. Chemical analysis by ¹H- and ¹³C-NMR, IR and GC-MS of KP12SA (methylated sample) showed 89.3 % of palmitic acid (C₁₆), 10.7 % of stearic acid (C₁₈) and traces of arachidic (C₂₀) and behenic acids (C₂₂). This study provides evidence that fatty acids present in Kalanchoe pinnata may be responsible, at least in part, for its immunosuppressive effect in vivo.

Introduction

The genus Kalanchoe (Crassulaceae) comprises about one hundred species, most of them native from Madagascar [1]. Kalanchoe pinnata (KP) is a perennial medicinal herb popularly used in Brazil and other parts of the world to treat various inflammatory diseases [2], [3], which contains substances such as bufadienolides [4], terpenoids and flavonoids [5]. In a previous work [6], we demonstrated that different extracts from KP leaves blocked human lymphocyte proliferation with no effect on the natural killer activity.

Treatment of mice with KP aqueous extract reduced the proliferative response of spleen cells to the mitogen and antigen; impaired the delayed-type hypersensitive reaction to ovoalbumin [3] and protected mice against progressive infection with Leishmania amazonensis [7], [8].

In this study, we describe the chemical composition of a potent fraction of Kalanchoe pinnata on lymphocyte proliferation.

Materials and Methods

General experimental procedures

Reverse phase column chromatographies were carried out on silanized silica gel RP-2 (70 - 230 mesh, Merck) using a gradient of water-methanol. A Sephadex LH-20 gel (25 - 100 mm, Sigma) column was also used. TLC plates (Silica 60 F₂₅₄ Merck; CH₂Cl₂ : MeOH, 98 : 2) were developed with a spray of ceric sulfate solution. ¹H- (200 MHz; DMSO-d ₆; δ 2.49 as internal reference) and ¹³C-NMR (50 MHz) spectra were recorded in a Varian Gemini 200.

Plant material

K. pinnata was collected during the autumn season, out of flowering time, at the garden of Universidade Federal do Rio de Janeiro campus (Brazil). A voucher specimen nr 292.697 is deposited at the herbarium of Rio de Janeiro's Botanical Garden.

Ethanolic extraction

Dried leaves (278 g), previously powdered, were macerated in ethanol at room temperature. The ethanolic extract (EE) was concentrated until dryness under vacuum at 65 - 70 °C, yielding 9 g of a dark-green syrupy material.

Purification of EE

The crude EE (9 g) was exhaustively washed with water at room temperature. The water-soluble fraction (EE-1; 4.3 g) was chromatographed on a RP-2 column yielding twelve fractions. The most T-cell suppressive fraction (KP12), eluted with MeOH, was further purified.

KP12 purification

KP12 (739 mg) was washed with CH₂Cl₂, yielding two fractions (KP12S and KP12I). The CH₂Cl₂-soluble fraction (KP12S; 625 mg) was chromatographed on a Sephadex LH-20 column (24 × 1.2 cm) with CH₂Cl₂ : EtOH (1 : 1) to afford only two fractions, KP12SA (532 mg) and KP12SB (14 mg). The most active fraction (KP12SA) was further analysed.

KP12SA: A light brown wax-like material; ¹H-NMR (DMSO-d ₆, ppm): δ = 0.93 (-CH_₃); 1.25 (intermediary chain CH_₂-); 1.50 (-CH_₂-CH₂-CO₂H); 2.20 (-CH_₂-CO₂H); ¹³C-NMR (DMSO-d ₆, ppm): δ = 174.5 (C-1), 33.7 (C-2), 31.3 - 24.5 (5 signals, C-3 to C-14), 22.1 (C-15), 13.9 (C-16). GC/MS data (Table [2]).

Methylation: Methylated samples from KP12SA (30 mg) and palmitic acid (Merck Lot 69370) were prepared as described by Schmitz and Pelka [9].

GC-MS: A HP6890 gas chromatograph using a HP5-MS capillary column (30 m × 0.25 mm I.D) was used for KP12SA analysis using palmitic acid as standard with injections of 1 μl for both samples (helium as carrier gas). Injector and detector temperatures were held at 270 °C. Column temperature was held at 70 °C for 1 min, and then programmed at a rate of 10 °C/min to 290 °C. Fatty acid methyl esters were identified by comparison with library standards data.

Methylated palmitic acid: GC/MS; Tetradecanoic acid methyl ester (C₁₅) t_R1 12.55 - 12.79 min (traces), m/z = 242 (M^+·), 199, 143, 87, 74, 55; 12-Methyltetradecanoic acid methyl ester (C₁₆) t_R2 13.56 - 13.70 min (traces), m/z = 256 (M^+·), 213, 143, 87, 74, 55; Palmitic acid methyl ester (C₁₇) t_R3 14.78 min (97.6 %), m/z = 270 (M^+·), 227, 143, 87, 74, 55; Heptadecanoic acid methyl ester (C₁₈) t_R4 15.21 min (traces), m/z = 284 (M^+·), 241, 185, 143, 87, 74, 55; Stearic acid methyl ester (C₁₉) t_R5 16.50 min (2.4 %), m/z = 298 (M^+·), 255, 143, 87, 74, 55.

Cells

BALB/c mice (20 g) lymphocytes from inguinal lymph nodes and human peripheral blood lymphocytes (PBMN) were prepared as described elsewhere [6], [7].

T Cell proliferation

Murine or human lymphocytes were cultivated as described elsewhere [6], [7]. Briefly: murine cells (4 × 10⁶ cells/ml) were cultivated with 2.5 μg/ml ConA [10] (concanavalin A, Sigma Chem. Co) 48 h at 37 °C in 5 % CO₂, in the absence or presence of different concentrations of crude extract or fractions. In control experiments, lymphocytes were cultivated in the presence of palmitic acid (Merck), cyclosporin A (Sandoz Pharmaceuticals, USA) or azathioprine (Microbiologica Química e Farmacêutica LTDA, Brazil). Proliferation was measured by incorporation of 0.5 μCi [³ H]-thymidine/well added 6 h before the end of the culture period. The cells were collected onto glass fibre filters using a dot-blot apparatus [11] and the incorporated radioactivity measured by liquid scintillation spectrometry. PBMN were cultured in the same way, except that the stimulus used was the human T cell mitogen phytohemagglutinin (PHA) at 5 μg/ml.

Results and Discussion

In this study we used the ethanolic extract (EE) obtained from leaves collected during the autumn season, in a region at sea level, where the plant grew under direct sunlight and with a low supply of water.

The fractionation of EE was guided by the murine and human T cell proliferation in vitro. Unprimed T cells are normally induced to proliferate by the lectins Con A and PHA [10]. These mitogens activate T cells by indirectly crosslinking their antigen receptors (TCR), and therefore serve well for monitoring T cell activation in a mixed cell population [10], [12]. Table [1] shows that EE was more potent to inhibit human lymphocyte proliferation (IC₅₀ = 17.5 μg/ml) than murine proliferation (IC₅₀ = 500 μg/ml). This different effect of EE may be due to the different stages of activation of these two kinds of cells.

The chromatography of the water-soluble fraction (EE-1), which corresponds to 47.8 % of EE, on a reverse-phase column resulted in twelve fractions. The most apolar of them (KP12) showed a very high inhibition of murine T cell proliferation (Table [1]). KP12, corresponding to 8.2 % of EE, was washed with CH₂Cl₂ yielding the soluble fraction KP12S which was much more active than the insoluble KP12I fraction on both murine and human cells. Further fractionation of KP12S on Sephadex LH-20 produced two subfractions. KP12SA which maintained its strong inhibitory activity and KP12SB, which was much less active, with an IC₅₀ higher than 50 μg/ml for both murine and human cells. The observation that fractionation of KP12S was not accompanied by further increase in its inhibitory activity may be due to the elimination of substances which acted in synergy with the most active substances concentrated in KP12SA.

The ¹H- and ¹³C-NMR analysis of KP12SA showed an aliphatic saturated chain. The triplet signal at 0.93 ppm (-CH_₃) corresponds to the terminal methyl group (-C_H₃; 13.6 ppm). The long intermediary proton methylene chain was assigned at 1.25 pm. The corresponding carbon signals were listed between 33.7 and 22.1 ppm.

The signal at 174.5 ppm was attributed to the carbonyl group of an acidic function. Methylene protons in the α and β positions to -COOH were listed at 2.20 (triplet) and 1.50 ppm (multiplet), respectively. No signals for olefinic carbons were observed, in agreement with the ¹H-NMR spectrum. The approached proton test (APT) ¹³C-NMR spectrum did not show any -CH signal, only confirming the -CH₃ group.

The fatty acid composition was also confirmed by the IR analysis. We observed a large band at 3400 cm^-1 attributed to the axial deformation of O-H. Bands between 2960 - 2870 cm^-1 correspond characteristically to saturated chains. The absorption at 1715 cm^-1 was attributed to the carbonyl of carboxyl group.

These experiments revealed that KP12SA could be a single or a mixture of two or more saturated fatty acids. In order to identify its composition, a sample was methylated and submitted to GC-MS. Two major signals were listed (Table [2]), one at t_R 14.63 min and other at t_R 16.52 min. The methyl esters corresponding to these peaks were identified by comparison of their retention time and mass spectrum fragmentation pattern with library standards data.

The major substance corresponds to 89.3 % of KP12SA (t_R 14.63 min) and was identified as palmitic acid (C₁₆). Stearic acid (C₁₈) was present at 10.7 % and corresponds to the signal at 16.52 min. Traces of two other saturated fatty acids were identified as arachidic (C₂₀) and behenic (C₂₂) acids.

Our data indicate that KP12SA, which is mainly constituted of palmitic acid, is a better inhibitor of lymphocyte proliferation than the crude extract, specially in regard to murine cells.

Many studies have investigated the level and composition of fat consumed in human and rat diet and its effects on the immune system [13], [14]. Studies showed that inhibitory effect of unsaturated fatty acids on human and murine lymphocytes [15] [16] [17] is more pronounced than that found with saturated fatty acids, such as palmitic and stearic acids. Our study provides, for the first time, evidence that an enriched saturated fatty acid fraction from Kalanchoe pinnata plays an important role on lymphocyte proliferation, which may explain its immunosuppressive effect in vivo.

Acknowledgements

We are indebted with Dr. T. Tomassini and J. C. R. de Albuquerque (FIOCRUZ, RJ, Brazil) for the GC-MS, Dr. J. Rabi (Microbiologica Química e Farmacêutica, Brazil) for the azathioprine gift and E. Miguez (NPPN, UFRJ, Brazil) for the NMR spectra. A. P. de Almeida thanks CNPq (Brazil) for her fellowship. Part of this work was supported by CNPq, FINEP (FNDCT 4389023900), FUJB, FAPERJ and CEPG (UFRJ, Brazil).

References

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  Search in Google Scholar
• 13 Yaqoob  P.. Monounsaturated fats and immune function.  Brazilian Journal of Medical and Biological Research. 1998;;  31 453-65
  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 16 Yaqoob  P,, Calder  P C.. The effects of fatty acids on lymphocyte functions.  Int. J. Biochem.. 1993;;  25 1705-14
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• 17 Fritsche  K L,, Byrge  M,, Feng  C.. Dietary omega-3 polyunsaturated fatty acids from fish oil reduce interleukin-12 and interferon-gamma production in mice.  Immunology Letters. 1999;;  65 167-73
  CrossrefPubMedSearch in Google Scholar

Dr. S. S. Costa*

Núcleo de Pesquisas de Produtos Naturais

Universidade Federal do Rio de Janeiro

21941-590, Rio de Janeiro

RJ, Brazil

Email: sscosta@nppn.ufrj.br

References

• 1 Allorge-Boiteau  L.. Madagascar Centre de Spéciation et d'Origine du Genre Kalanchoe (Crassulaceae).  Biogéographie de Madagascar. Lourenço WR, Editions de l'ORSTOM, Paris; 1996: 137-45
  Search in Google Scholar
• 2 Hema  D,, Tidjani  M,, Bassene  E,, Pousset  J L,, Giono-Barber  H.. Plantes Médicinales Africaines XXXIV Étude de l'action anti-inflammatoire de Bryophyllum pinnatum (Crassulacées).  Plantes Médicinales et Phytothérapie. 1986;;  XX 231-35
  PubMedSearch in Google Scholar
• 3 Rossi-Bergmann  B,, Costa  S S,, Borges  M BS,, Da Silva  S AG,, Noleto  G R,, Souza  M LM,, Moraes  V LG.. Immunosuppressive effect of the aqueous extract of Kalanchoe pinnata in mice.  Phytotherapy Research. 1994;;  8 399-02
  PubMedSearch in Google Scholar
• 4 Yamagishi  T,, Haruna  M,, Yan  X-Z,, Chang  J J,, Lee  K H.. Antitumor agents, 110. Bryophyllin B, a novel potent cytotoxic bufadienolide from Bryophyllum pinnatum.  Journal of Natural Products. 1989;;  52 1071-79
  PubMedSearch in Google Scholar
• 5 Costa  S S,, Jossang  A,, Bodo  B.. Propriétés biologiques et phytochimie des Kalanchoe.  Kalanchoe (Crassulacées) de Madagascar, Systématique, écophysiologie et phytochimie,. Boiteau P, Allorge-Boiteau L Karthala, Paris; 1995: 219-35
  Search in Google Scholar
• 6 Moraes  V LG,, Santos  L FM,, Castro  S B,, Loureiro  L H,, Lima  O A,, Souza  M LM,, Yien  L MK,, Rossi-Bergmann  B,, Costa  S S.. Inhibition of lymphocyte activation by extracts and fractions of Kalanchoe, Alternanthera, Paullinia and Mikania species.  Phytomedicine. 1994;;  1 199-04
  PubMedSearch in Google Scholar
• 7 Da Silva  S AG,, Costa  S S,, Mendonça  S CF,, Silva  E M,, Moraes  V LG,, Rossi-Bergmann  B.. Therapeutic effect of oral Kalanchoe pinnata leaf extract in murine leishmaniasis.  Acta Tropica. 1995;;  60 201-10
  CrossrefPubMedSearch in Google Scholar
• 8 Da Silva  S AG,, Costa  S S,, Rossi-Bergmann  B.. The antileishmanial effect of Kalanchoe is mediated by nitric oxide intermediates.  Parasitology. 1999;;  118 575-82
  CrossrefPubMedSearch in Google Scholar
• 9 Schmitz  A A,, Pelka  J R.. Rapid preparation of fatty acids esters from lipids for gas chromatographic analysis.  Analytical Chemistry. 1966;;  38 514-15
  CrossrefPubMedSearch in Google Scholar
• 10 Andersson  J,, Moller  G,, Sjoberg  O.. Selective induction of DNA synthesis in T and B lymphocytes.  Cellular Immunology. 1972;;  4 381-93
  CrossrefPubMedSearch in Google Scholar
• 11 Rossi-Bergmann  B,, Noleto  G R.. A low-cost and efficient procedure for harvesting DNA-labelled cells using a dot-blot apparatus.  Biotechniques. 1994;;  17 678-81
  PubMedSearch in Google Scholar
• 12 Kruisbeek  A M,, Shevach  E.. Proliferative assays for T cell function.  Current Protocols in Immunology. John Wiley and Sons, 1995; Vol I: 3-12
  Search in Google Scholar
• 13 Yaqoob  P.. Monounsaturated fats and immune function.  Brazilian Journal of Medical and Biological Research. 1998;;  31 453-65
  PubMedSearch in Google Scholar
• 14 Calder  P C.. Fat chance of immunomodulation.  Immunology Today. 1998;;  19 244-47
  CrossrefPubMedSearch in Google Scholar
• 15 Jeffery  N M,, Cortina  M,, Newsholme  E A,, Calder  P C.. Effects of variations in the proportions of saturated, monounsaturated and polyunsaturated fatty acids in the rat diet on spleen lymphocyte functions.  British Journal of Nutrition. 1997;;  77 805-23
  PubMedSearch in Google Scholar
• 16 Yaqoob  P,, Calder  P C.. The effects of fatty acids on lymphocyte functions.  Int. J. Biochem.. 1993;;  25 1705-14
  PubMedSearch in Google Scholar
• 17 Fritsche  K L,, Byrge  M,, Feng  C.. Dietary omega-3 polyunsaturated fatty acids from fish oil reduce interleukin-12 and interferon-gamma production in mice.  Immunology Letters. 1999;;  65 167-73
  CrossrefPubMedSearch in Google Scholar

Dr. S. S. Costa*

Núcleo de Pesquisas de Produtos Naturais

Universidade Federal do Rio de Janeiro

21941-590, Rio de Janeiro

RJ, Brazil

Email: sscosta@nppn.ufrj.br