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Biomedicine & Preventive Nutrition 3 (2013) 151–157
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Original article
Immunomodulatory activity of isoflavones isolated from Iris kashmiriana: Effect on T-lymphocyte proliferation and cytokine production in Balb/c mice Nighat Nazir Department of Chemistry, Islamia College of Science and Commerce, Hawal, Srinagar 190002, J&K, India
a r t i c l e
i n f o
Article history: Received 9 November 2012 Accepted 12 December 2012 Keywords: Iris kashmiriana Iridaceae Isoflavones Cytokines Immunomodulators Flowcytometry
a b s t r a c t This study deals with the evaluation of immunomodulatory action of a new isoflavone isonigricin (1) and first report isoirisolidone (2) isolated from rhizomes of Iris kashmiriana. Flowcytometric method was employed to study their effect on production of T-lymphocytes (CD4+ and CD8+ T-cells) and T-cell cytokines (IL-2, IL-4 and IFN-␥) in a dose-dependant manner. Drugs at doses of 0.025 to 0.8 mg/kg per oral dose showed compound 1 to possess immunosuppressant activity on T-cells as well as cytokines, while compound 2 acted as immunostimulator for both cells and cytokines. However, their methylated products viz. 1OMe and 2OMe exhibited stimulatory effect on interleukins under study and suppressive effect on production of T-lymphocytes and IFN-␥. The oral LD50 in mice was observed to be more than 200 mg/kg body weight. Such a difference in their activity is the manifestation of the position and nature of substituents present on isoflavonoidal skeleton and demonstrates the potential of these isolates as prospective immunomodulatory agents. © 2013 Published by Elsevier Masson SAS.
1. Introduction The immune system is a highly complex and extraordinarily sophisticated network involving proteins, special cells, tissues and organs that work together to defend the body against invasion by foreign individuals like germs, parasites, toxins etc. Thus, it acts as the body’s defence against infections that works through a series of steps called the immune response. Prominent immuno factors of antigen-specific immune responses are T-lymphocytes (including T-helper cells, T-suppressor cells and cytotoxic T-cells) and their cytokines. The healthy state is believed to be based on a fine-tuning of various humoral and cellular factors functioning in the immunoregulatory mechanism. Any sort of imbalance in the production or expression of these immune factors can result in a pathological condition. A number of factors like food, drugs, pollutants, physical and psychological stress, hormones etc. can influence this balance, resulting in either immunostimulation or immunosuppression. Such conditions have been shown to be involved in the etiology as well as pathophysiology of many diseases of skin, gut, respiratory tract, joints and central organs [1–4]. The role of cytokines in the pathogenesis of autoimmune diseases has been the subject of intense study in recent years. For example, type-1 cytokines (IL-2, IFN-␥, TNF-␣) are involved in autoimmune diseases such as type-1 diabetes, multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases and acute graft versus host
E-mail address: [email protected] 2210-5239/$ – see front matter © 2013 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.bionut.2012.12.006
disease. On the contrary, type-2 cytokines (like IL-4, IL-5, IL-12) are associated in allergic response and inhibit cell-mediated immunity [5,6]. Modulation of immune response through stimulation or suppression may help in maintaining a disease-free state [7]. Modification of immune function by pharmacological agents is emerging as a major area of therapeutics. In recent years, there has been great interest in the pharmacological manipulation of the immune response. Selective pharmacological action on an individual component of a complex immune response is seen as a particularly attractive approach to the therapy of immunologically mediated diseases. Immunomodulatory activity is a collective term indicating biological or pharmacological effect on various factors functioning in the immune response which requires timely interaction of multiple cell types and its modulation aims to restore immune homeostasis. Immunomodulators are the agents that help in restoring a proper balance of these immune factors and are effective for treating and preventing diseases and illnesses that stem from such imbalances. Immunomodulators can be either stimulators or suppressors. Former are helpful in preventing immunodeficiencies or for generalized immunosuppression following drug treatment, for combination therapy with antibiotics, and as adjuvants for vaccines and other depressed states of immunity. Those metabolites that suppress immune reactions are potentially useful to mitigate autoimmune diseases such as systemic lupus erythmatosus, hypersensitivity reactions, graft rejection or certain gastrointestinal tract diseases. Presently available immunomodulatory drug have side effects such as hypertension, renal failure, oncogenicity, hypertrichosis [8], myelosuppression [9], anemia [10] and are
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expensive [8]. Moreover, constant increase in antibiotic resistant strains of microorganisms has prompted scientists to look for herbal immunomodulators to treat various infections. Modulation of immune functions using medicinal plants and their products as a possible therapeutic measure has become a fundamental principle of therapeutic approach. Flavonoids are prominent plant secondary metabolites with variable phenolic structures and are consumed by humans as dietary constituents. Although not considered nutrients and essential for life, flavonoid ingestion may play a significant role in health and disease [11,12]. Isoflavones is a large subclass of flavonoids with 15-carbon (C6-C3-C6) backbone. Numerous studies have shown an association between isoflavone rich dietary consumption and reduced cancer risk, particularly breast and prostrate cancer [13–15]. It has been shown that some isoflavones can act as inhibitors of multidrug resistance transporter MRP1 through influencing the biophysical properties of membranes [16]. The preventive role of isoflavones in cancer, cardiovascular diseases, osteoporosis, obesity and menopausal symptoms [13,17–19], in addition to their antioxidant [20,21], antimicrobial [22], antiinflammatory and estrogenic activities [14,15] has been largely documented. The estrogenic activity of genistein, daidzen and equol are currently being extensively investigated at the molecular, pre-clinical and clinical levels to determine their potential for treatment of chronic diseases such as hormone-dependent cancer, cardiovascular diseases and osteoporosis [23]. Flavonoids have also been reported to show immunomodulatory properties by affecting varied cells of immune system. Recent reports [24,25] indicate that several types of flavonoids stimulate human peripheral blood leukocyte proliferation. They significantly increase the activity of helper T-cells, cytokines, interleukin 2 (IL-2), interferon ␥ (IFN-␥) and macrophages and are thereby useful in the treatment of several diseases caused by immune system dysfunction. Luteolin, 3 ,4 ,5,7tetrahydroxyflavone, an important member of the flavonoid family has shown to exert immunomodulatory effects that may be beneficial in the treatment of neurodegenerative diseases such as multiple sclerosis, which has an underlying T-cell mediated autoimmune pathology [24,25]. We have previously reported the immunomodulatory effect of two isoflavones, irilone and irisolidone on the production of T-lymphocytes (CD4+ and CD8+) and T-cell cytokines namely IL-2, IFN-␥, IL-4 and IL-5 that was found to be dependent on the position and nature of substitution [26]. However, methylation of these compounds drastically decreased their activities. The wide spectrum of pharmacological activities associated with the isoflavones and the growing interest in investigating natural isolates for possible T-cell modulating properties, prompted us to undertake the chemical investigation of Iris kashmiriana (reported to be a rich source of isoflavones) [27,28] and then screen the pure isolates for their immunomodulatory activity. Therefore, the present report deals with the evaluation of immunomodulatory activity of new isoflavone isonigricin (1) and a first report isoirisolidone (2) isolated previously [28] from the rhizomes of I. kashmiriana and their O-methylated products (1OMe and 2OMe) with respect to T-lymphocytes (CD4+ and CD8+ cells) and T-cell cytokine (IL-2, IL-4 and IFN-␥) production in mice using flowcytometry. The results presented merely point to the effect of the isoflavones under study on the production of T-lymphocytes and cytokines and hence to their potential prospective clinical application as immunomodulatory agents. 2. Materials and methods 2.1. General Melting points were determined in one end open capillaries on Buchi 570 melting point apparatus and are uncorrected. IR spectra
were determined in KBr pellets on Perkin–Elmer spectrometer. 1 H and 13 C-NMR were recorded on a Bruker 200 MHz spectrometer using TMS as internal standard. Mass spectra were determined on Jeol-MSD300 and Bruker Esquire 3000. Column chromatography was carried out on Silica-gel (60–120 mesh size, E. Merck). TLCs were run on 0.25 mm silica gel 60 F254 plates using UV light or Ceric ammonium sulfate solution for detection of the spots. Fluorisothiocyanate (FITC)–labeled CD4 anti-mouse monoclonal antibody, Phycoerythrin (PE)–labeled CD8 anti-mouse monoclonal antibody, FACS lysin solution (BD Bioscience, USA), Levamisole, cyclosporinA (Sigma-Aldrich, India), wash buffer, mouse cytokine standards (IL-2, IFN-␥, IL-4, IL-5) (BD Biosciences, USA) were used. Unless and otherwise specified, all the solvents were of analytical grade (Qualigens). 2.2. Plant material The rhizomes of I. kashmiriana were purchased from Nehru Botanical Garden, Srinagar (J&K, India), identified at Institute of Plant Taxonomy, University of Kashmir, under Acc. No. 13595 and Collection No. 1207 GH DAR. 2.3. Extraction and isolation Air dried and powdered rhizomes of the plant (1.5 kg) were extracted with Petroleum ether (4 l, 60–80 ◦ C, Soxhlet, 48 hours) and the extract concentrated and reduced to 250 ml volume and left overnight at room temperature (20–22 ◦ C) when a pale yellow solid precipitated out and was filtered. The crude material was dissolved in a mixture of chloroform:hexane (7:3) and left at 0 ◦ C to give a crystalline solid which on filtration and recrystallisation in the same solvent system afforded fine needles of compound 2 (138 mg, 0.0092% yield, mp 220 ◦ C). The defatted rhizomes were extracted with methanol (4 l, soxhlet, 36 hours) and the extract de-solventised and lyophilized (using Christ Alpha 1-4 lyophiliser) to give dried material (68 g). Chromatography of the crude extract (60 g) was carried out on silica gel (60–120 mesh) using an increasing gradient of n-hexane:chloroform followed by increasing gradient of chloroform:methanol mixture. Fractions of 200 ml volume each were collected, analyzed by TLC and pooled accordingly. Fractions 25-30 eluted with 1:3n-hexane:chloroform, pooled, concentrated and rechromatographed in n-hexane: chloroform yielded 93 mg of 2. Fractions 57-61 (95:5 chloroform:methanol) were combined and concentrated to yield a pale yellow amorphous powder (430 mg) which on dissolution in methanol (50 ml, reflux conditions) followed by cooling afforded colorless shiny crystals of 1 (355 mg, 0.023% yield, mp 249 ◦ C). 2.4. Preparation of methyl derivatives of compound 1 and 2 (1OMe and 2OMe) To a methanolic solution of compound 1 (100 mg, 0.32 mmol, 50 ml), was added an ethereal solution of diazomethane (50 ml) (prepared from 5 g of nitrosomethyl urea) and the contents were left at 10 ◦ C for 8 hrs. The contents were concentrated and the residue column chromatographed on silica gel (60–120 mesh size) and eluted with 10% ethyl acetate in n-hexane to give 1OMe – a white solid (76 mg, 0.23 mmol, 71.8% yield). The compound 2OMe was prepared from compound 2 (80 mg, 0.25 mmol) by the procedure as described above (63 mg, 0.184 mmol, 73.6% yield). 2.5. Spectral data 2.5.1. Spectral data of compound 1 Colorless solid analysed for C17 H12 O6 (Cal. C 65.38 H 3.873% found C 65.98 H 3.941%); mp 249 ◦ C; UV (MeOH): Max 213, 264
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& 322 nm; IR (KBr): max = 3267, 2952, 2914, 1632, 1591, 1516, 1499, 1467, 1439, 1371, 1343, 1287, 1259, 1215, 1172, 1128, 1104, 1079, 1057, 1036, 946, 926, 847, 835, 823, 755, 714, 666 cm−1 ; 1 HNMR (200 MHz, DMSO-d6 )␦(ppm) = 3.92 (3H, s, OMe), 6.12 (2H, s, -OCH2 O-), 6.81 (2H, d, J = 8.43 Hz, 3 ,5 -H), 7.02 (1H, s, 5-H), 7.36 (2H, d, J = 8.41 Hz, 2 , 6 -H), 8.20 (1H, s, -OCH C); 13 C-NMR (125 MHz, DMSO-d6 )␦(ppm) = 152.53, 122.37 174.09, 103.54, 135.86, 153.93, 140.41, 151.01, 1133.16, 102.58, 124.20, 130.22, 114.83, 157.06, 114.83, 130.22, 60.77; MS: m/z (%): 312 (100), 297 (100), 244 (50), 219 (23), 194 (36), 152 (37), 146 (12), 118 (7) and 93 (9). 2.5.2. Spectral data of compound 1OMe A white solid; mp 144–45 ◦ C; IR (KBr): max = 2964, 2920, 1644, 1605, 1508, 1473, 1432, 1393, 1356, 1308, 1285, 1257, 1241, 1215, 1175, 1099, 1077, 1053, 1035, 945, 925, 913, 852 cm−1 ; 1 H-NMR (500 MHz, CDCl3 )␦(ppm) = 3.84 (3H, s, OMe), 4.08 (3H, s, OMe), 6.07 (2H, s, -OCH2 O-), 6.63 (1H, s, 5-H), 6.93 (2H, d, J = 8.6 Hz, 3 ,5 -H), 7.12 (2H, d, J = 8.6 Hz, 2 ,6 -H), 7.68 (1H, s, -OCH C); 13 CNMR (50 MHz, CDCl3 )␦(ppm) = 55.32, 61.25, 93.24, 102.16, 113.82, 113.88, 124.13, 125.33, 130.37, 135.57, 135.81, 140.70, 150.23, 152.78, 154.72, 154.80, 159.51, 175.46; MS: m/z (%): 326 (84) 298 (77), 297 (100), 280 (74), 252 (49), 194 (34), 164 (42), 151 (29), 132 (88), 93 (23), 89 (51). 2.5.3. Spectral data of compound 2 A pale yellow crystalline solid analyzed for C17 H14 O6 (Cal. C 64.97 H 4.89% found C 65.71 H 4. 52%); mp 220 ◦ C; UV (MeOH): Max 213, 265 & 340 nm (+AlCl3 : 237, 269, 312, 364 nm, +AlCl3 + HCl 233, 274, 312, 362 nm, NaOAc 222, 273, 341 nm, NaOAc + H3 BO3 221, 267 nm); IR (KBr): max = 3438, 3068, 2989, 2959, 1660, 1624 cm−1 ; 1 H-NMR (500 MHz, CDCl )␦(ppm) = 3.85 & 4.04 (3H each, s, 2x3 OMe), 6.53 (1H, s, 8-H), 6.99 (2H, dd, J = 2.0 & 8.74 Hz, -3 ,5 -H), 7.46 (2H, dd, J = 2.0 & 8.71 Hz, 2 ,6 -H), 7.88 (1H, s, -OCH C); 13 C-NMR (125 MHz, CDCl3 )␦(ppm) = 155.16, 122.89, 181.28, 152.64, 130.37, 153.47, 93.21, 152.83, 106.46, 123.12, 130.15, 114.12, 159.80, 114.12, 130.15, 55.37, 56.89; MS: m/z (%): 314 (100), 299 (50), 252 (7), 207 (52), 203 (11), 192 (6) & 164 (12). 2.5.4. Spectral data of compound 2OMe A white solid; mp180 ◦ C; IR (KBr): max = 2947, 2923, 1642, 1606, cm−1 ; 1 H-NMR (200 MHz, CDCl3 )␦(ppm) = 3.88, 3.96, 3.98 & 4.01 (12H, s, 4x-OMe), 6.65 (1H, s, 8-H), 6.98 (2H, d, J = 8.8 Hz, 3 ,5 -H), 7.43 (2H, d, J = 8.8 Hz, 2 ,6 -H); 13 C-NMR (50 MHz, CDCl3 )␦(ppm) = 53.30, 56.20, 61.50, 62.00, 94.01, 105.72, 114.18, 122.73, 123.04, 130.13, 129.86, 152.31, 153.24, 154.56, 155.75, 159.70, 175.30; MS: m/z (%): 342 (34), 327 (88), 312 (100), 299 (11), 195 (17), 182 (9), 154 (14), 132 (15), 124 (17), 122 (29). 2.6. Animals and drug Eight to 10-week-old Balb/C mice (Mus musculus), 18–22 g body weight in groups of six were employed for the study. The animals were housed under standard conditions of temperature (23 ± 2 ◦ C), relative humidity (55 ± 10%), 12 hours photoperiod and fed with standard pellet diet (Lipton India Ltd) and water ad libitum. In every experiment, one group of animals was used as a sensitized vehicle control and another as a non-sensitized naïve control. Drugs were freshly prepared as a homogenized suspension in 1% w/v acacia gum and administered orally daily once a day for the duration of experiment. Cyclosporin (immunosupressant) and Levamisole (immunostimulant) were used as standard drugs and were administered once daily at 5 mg/kg and 2.5 mg/kg per oral dose respectively. The treated groups received drugs at 0.025, 0.05, 0.10, 0.20, 0.40 and 0.80 mg/kg per oral dose.
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2.7. Antigen One hundred microliter of ovalbumin (2 mg/ml PBS) was injected intraperitonally in experimental mice for immunization. Drugs were administered for seven days including the day of immunization. On eighth day, animals were challenged by injecting same amount of ovalbumin i.p to induce the challenge. 2.8. Acute Toxicity Test Acute Oral Toxicity studies were carried out following OECD Guidelines no. 423. After a single dose treatment with drug extract (200 mg/kg per oral dose) to respective groups (female mice only), the animals were observed continuously for 1 hour and then half hourly for 5 hours for any change in general behavior and finally up to 15 days for any mortality. 2.9. Study design for CD4+ and CD8+ T cell estimation One hundred microliter of whole blood, with 10 l of Mab (monoclonal antibody) FITC (fluorescein isothiocyanate) anti-CD4 and 10 l of Mab (monoclonal antibody) PE (Phycoerythrin) antiCD8 were added and mixed gently. Tubes were incubated in dark for 30 minutes at room temperature. Samples were again incubated at room temperature for 10 minutes after adding 2 ml of 1X FACS lyses solution (BD Biosciences). Samples were spinned (300–400Xg) for 10 minutes at room temperature. The supernatant was aspirated and sample was given three washings of phosphate buffer saline (pH). The resulting stained cell pellet was resuspended in 500 l of phosphate buffer saline and was run on a flow cytometer (LSR, BD Biosciences). Acquisition and the analysis were done using Cell Quest Pro software (BD Biosciences). 2.10. Study design for IL-2, IFN- (Th1) and IL-4 (Th2) cytokine estimation To detect the intracellular expression of cytokines, anti-CD4FITC, anti-interleukin (IL-2) PE, anti-interleukin (IL-4) PE, and anti-CD8 PE, anti-interferon (IFN-␥) FITC was analysed. A total of 100 l of whole blood was incubated with 10 l of Mab (monoclonal antibody) FITC-anti-CD4 in the dark incubation for 30 min at room temperature. Erythrocytes were lysed for 10 min incubation at room temperature with 2 ml 1X FACS Lysing solution (Becton Dickinson) and centrifugation at 300Xg for 10 min. After the cells were washed once with 1 ml phosphate buffer solution (PBS)/0.5% BSA by centrifugation at 300Xg for 5 min. Supernatants were removed and 500 l of permeabilization solution and 10 l of each monoclonal antibody, anti-IFN-␥ FITC, anti-IL-2 PE and anti IL-4 PE was added to separate the tubes containing the cell pellets. The samples were votexed and incubated for another 30 min at room temperature. The permeabilized cells were finally washed in 1 ml PBS/0.5% BSA by centrifugation at 300Xg for 5 minutes. Supernatants were removed and the cells were resuspended in 500 l 1% PFA, and stored at 2–8 ◦ C in the dark. The fixed cells were then analyzed within 24 h in a flow cytometric analyzer using Cell Quest Pro software (BD Biosciences). 2.11. Statistical analysis The significance of differences between control and treated groups was analyzed using Student’s t-test. Differences were considered significant at P ≤ 0.01 and significant values are represented by asterisk marks. All experiments were repeated at least three times. Data are expressed as % mean ± SE.
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OMe O
O
CD4+
(a)
1 1OMe 2 2OMe
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O
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O
O
MeO
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(%) Mean±SE
OH
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OH
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HO
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OMe
Compounds 1 and 2 were identified as isonigricin and isoirisolidone respectively (Fig. 1) on the basis of their physical and spectral analyses like IR, NMR and mass spectrometry as previously reported by us [28].
0.8 mg/kg
0.400 mg/kg
0.200 mg/kg
0.100 mg/kg
0.050 mg/kg
0.025 mg/kg
Cyclosporine 5mg/kg
3.1. Identification of isoflavones
Cont. (Sensitised)
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0
3. Results
Levamisole 2.5mg/kg
Fig. 1. Structure of compound (a) 1 and (b) 2.
Fig. 2. Percentage age change in CD4+ cell expression in Balb/c mice treated with different concentrations of 1, 1OMe, 2 and 2OMe. Results are expressed as mean ± SE for group of six mice, and are representative of these experiments. Statistical significance, * P < 0.01, ** P < 0.001 when compared to control (sensitized) mice.
3.2. Effect on general behavior and acute toxicity
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CD8+
1 1OMe 2 2OMe
**
20
**
3.4. Effect on Th1 cytokines (IFN- and IL-2) and Th2 cytokine (IL-4) Figs. 4–6 show dose dependent effect of 1, 2, 1OMe and 2OMe at doses 0.025 to 0.800 mg/kg per oral dose, on IL-2, IFN-␥ and IL-4 cytokine expression respectively. The results show significant suppressive effect of 1 (close to suppressive effect of standard drug cyclosporin), being maximum at 0.40 mg/kg dose level, while
** ** **
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* *
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0.400 mg/kg
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Dose dependent effect of isoflavone1, 2, 1OMe and 2OMe at doses 0.025 to 0.800 mg/kg per oral dose, on CD4+ and CD8+ T-cells using flow cytometry are presented in Figs. 2 and 3 respectively. Animals treated with isoflavone1 and 2 showed suppression and stimulation respectively in CD4+ and CD8+ T-cells expression in comparison with sensitized control. The maximum suppressive effect of 1 was observed at a dose level of 0.80 mg/kg where it was 20.38% while maximum stimulatory effect of 21.93% for 2 was observed at a dose level of 0.05 mg/kg being even more than standard drug Levamisole (18.01%) at 2.5 mg/kg dose level in case of CD8+ T-cells. Methylated product of 1 (1OMe) again showed suppressive effect on CD4+ T-cell expression but for CD8+ T-cell expression, it showed stimulatory effect with maximum activity at dose level of 0.025 mg/kg. On the other hand, 2OMe exhibited suppress give effect on CD4+ and CD8+ cells contrary to 2. The maximum suppression by 2OMe was at dose level of 0.025 mg/kg that was comparable to immunosuppressive standard drug cyclosporin at dose level of 2.5 mg/kg.
(%) Mean±SE
**
0.200 mg/kg
3.3. Effect on CD4+ and CD8+ T cells
0.8 mg/kg
A single dose of drugs administered in each group of mice did not show any change in general behavior of the test animals. No mortality was observed over a period of 15 days up to the dose level of 200 mg/kg orally. So acute LD50 was found to be greater than 200 mg/kg.
Fig. 3. Percentage age change in CD8+ cell expression in Balb/c mice treated with different concentrations of 1, 1OMe, 2 and 2OMe. Results are expressed as mean ± SE for group of six mice, and are representative of these experiments. Statistical significance, * P < 0.01, ** P < 0.001 when compared to control (sensitized) mice.
stimulatory effect of 2 (more than standard drug Levamisole for IL-4 cytokine expression), being maximum at 0.05 mg/kg of dose level. 1OMe, contrary to 1, showed stimulatory effect on IL-2 and IL-4 cytokine expression with maximum effect at 0.05 mg/kg of dose level while suppressive effect on IFN-␥ being maximum at
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IL-2
40
1 1OMe 2 2OMe
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IL-4
1 1OMe 2 2OMe
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*
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*
**
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Levamisole 2.5mg/kg
Fig. 6. Percentage age change in IL-4 expression in Balb/c mice treated with different concentrations of 1, 1OMe, 2 and 2OMe. Results are expressed as mean ± SE for group of six mice, and are representative of these experiments. Statistical significance, * P < 0.01, ** P < 0.001 when compared to control (sensitized) mice.
4. Discussion
50
IFN-γ
1 1OMe 2 2OMe
40 ** **
(%) Mean±SE
Cont. (Sensitised)
Cont. (Naïve)
0.8 mg/kg
0.400 mg/kg
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0.050 mg/kg
0.025 mg/kg
Cyclosporine 5mg/kg
Levamisole 2.5mg/kg
Cont. (Sensitised)
Cont. (Naïve)
Fig. 4. Percentage age change in IL-2 expression in Balb/c mice treated with different concentrations of 1, 1OMe, 2 and 2OMe. Results are expressed as mean ± SE for group of six mice, and are representative of these experiments. Statistical significance, * P < 0.01, ** P < 0.001 when compared to control (sensitized) mice.
**
**
**
0
Cyclosporine 5mg/kg
0
**
** **
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**
**
**
0.8 mg/kg
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**
0.050 mg/kg
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0.400 mg/kg
**
0.025 mg/kg
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0.8 mg/kg
0.400 mg/kg
0.200 mg/kg
0.100 mg/kg
0.050 mg/kg
0.025 mg/kg
Cyclosporine 5mg/kg
Levamisole 2.5mg/kg
Cont. (Sensitised)
Cont. (Naïve)
0
Fig. 5. Percentage age change in IFN-␥ expression in Balb/c mice treated with different concentrations of 1, 1OMe, 2 and 2OMe. Results are expressed as mean ± SE for group of six mice, and are representative of these experiments. Statistical significance, * P < 0.01, ** P < 0.001 when compared to control (sensitized) mice.
0.80 mg/kg of dose level. 2OMe also showed stimulation of IL-2 and IL-4 cytokine expression at higher dose levels in tune with 2 but a suppressive effect on IFN-␥ with maximum effect at 0.025 mg/kg dose level.
Modulation of immune response through stimulation or suppression may help in maintaining a disease-free state. Immunopharmacology is a comparatively new and developing branch of pharmacology aiming at searching immunomodulating agents with no or not very serious side effects. There is, thus, a growing interest in identifying and characterizing natural compounds with immunomodulatory activity. Our results show the immunomodulating activity of isoflavones 1 and 2, indicating their possible role in treating pathophysiological conditions involving immunological background. The compound 1 induced a doserelated inhibition of CD4+ and CD8+ T-cell expression (Figs. 2 and 3) and cytokine IL-2, IFN-␥ and IL-4 production (Figs. 4–6). T-cells expressing CD4 are increased when there is a general expansion due to increased immunological activity of T-cells. Thus an inhibition of this expression by 1 in a dose-dependent manner is an indication of its immunosuppressive activity. Due to its effect on T-cells and B-cells, IL-2 is a central regulator of immune responses. Under physiological conditions, IL-2 is produced mainly by T-cells expressing the surface antigen CD4 following cell activation by mitogens or allogens. Thus suppression of CD4+ T-cells inhibits IL-2 presence and is also responsible for inhibition of IL-4 production by CD4+ T-cells. Since IL-2 functions to stimulate the synthesis of IFN-␥ by T-lymphocytes and natural killer (NK) cells and also stimulates the differentiation of naïve CD4 T-cells into IFN-␥ producing Th-1 cells, the inhibition of IL-2 is possibly responsible for reduced IFN-␥ secretion by Th-1 type CD4+ and CD8+ cells. These findings demonstrate that 1 has a potent T-cell and cytokine suppressive activity and suggestive of its possible therapeutic usefulness, especially to mitigate autoimmune or certain gastrointestinal tract diseases and most importantly against allograft rejection which has been shown to be associated with an increase in IL-2 and other pro-inflammatory cytokines such as IFN␥ and TNF-␣. The most effective transplantation immunosupressive strategies are based on interruption of IL-2 signalling by cyclosporin
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A and Tacrolimus. However, treatment with these drugs is associated with serious adverse side effects including nephrotoxic, diabetogenic, neurological and cardiovascular effect [29]. Apparent safety over long-term administration of 1 is encouraging enough to warrant further studies to explore its possible therapeutic role in modern clinical practice. Dose dependent potentiating effect of 2 on CD4+ and CD8+ Tcells (Figs. 2 and 3) along with the increase of IL-2, IFN-␥ and IL-4 (Figs. 4–6) secretion shows its immunostimulatory effect, being even more than standard drug levamisole in case of CD8+ T-cells. The LD50 of 2 has been shown to be greater than 200 mg/kg body weight, its stimulatory effect at a dose level as small as 0.800 mg/kg body weight in comparison to the dose level of 2.5 mg/kg body weight of levamisole suggests it as a potential immunostimulant. Moreover, adverse side effects have been attributed to levamisole. These are agranulocytosis – leaving patients susceptible to fulminate and opportunistic infections and can also cause debilitating cutaneous necrotizing vasculitis [30,31], multifocal inflammatory leukoencephalopathy [32,33] and it could be a triggering factor in multiple sclerosis [34]. This suggests the possible usefulness of 2 as immunomodulatory agent for treatment of cancer, immunodeficiency diseases, or for general immunosupression following drug treatment, for combination therapy with antibodies and as adjuvants for vaccines. The immunosuppressive effect 1 and immunostimulatory effect of 2 could be apparently attributed to the nature of the functional groups present in the two compounds as the basic nucleus in both the compounds is same i.e. an isoflavone nucleus. In order to explore the possibility that the functional groups in ring A and B of 1 and 2 could be responsible for the said effect, an examination of the structure of 1 and 2 shows a free phenolic group at 4 position in ring B of 1 which is present as OMe group in compound 2. In ring A of compound 1, no free phenolic groups is present, which however is present in compound 2 showing thereby higher pka in ring A of 1 compared to that of ring A of 2. Interestingly, three positions in ring A of both the compounds are substituted. Two possibilities could be comptemplated namely i) the substituents in the ring A or ii) 4 substituent in ring B responsible for the antagonistic effect. We, therefore, decided to methylate compounds 1 and 2 and subject them to flow cytometric studies and study the effect of decrease of the charges in the ring A and ring B. The results obtained showed that the methylation of 4 in ring B of compound 1 again had a suppressive effect on the CD4+, CD8+ T-cells and IFN␥ (Figs. 2, 3 and 5) but had a stimulating effect on IL-2 and IL-4 which increases with an increase in dose level. Thus, it is in contrary to its 4’-demethylated product 1 (Figs. 4 and 6). In case of compound 2, there was significant change in activity after methylation. A decrease in the stimulatory effect could be seen for IL-2 and IL-4 while as the values of CD4+ and CD8+ T-cells and IFN␥ shifted towards suppression at several dose levels. Figs. 2 to 6 show the %age increase/decrease of the immunomodulating activities of the parent compounds and their methylated derivatives. The results are suggesting that free hydroxyls in ring A of isoflavones can be responsible for overall stimulatory activity of 2 whereas free hydroxyls in ring B can cause overall suppressive activity of 1. Switching over of suppressive effect to stimulation for IL-2 and IL-4 by methylation of 4 hydroxyl group of 1 in tune with stimulatory effect of 2 and 2OMe (both having methylated 4 position) for IL-2 and IL-4, suggest that expression of these two immune factors is favoured by methylated substitution in ring B. In the same manner changing of stimulatory effect of 2 for IFN-␥, CD4+ and CD8+ T-cells by methylation of ring A hydroxyls to suppressive effect, that is also observed with 1 and 1OMe suggests that free hydroxyl substitution in ring A could be a contributing factor for observed enhanced stimulation of these immunofactors. Thus, it could be concluded that the nature of substituents in ring A or in
other terms the pKa in ring A may be responsible for the observed suppressant or stimulation effect. It may be said that, there could be other factors like the position of the functional groups and their nature in the ring A which may also be responsible for the said effect. These findings are in quite agreement with the earlier studies where the importance of position and number of phenolic substitution on isoflavone backbone for immunomodulatory activity has been discussed [26,35]. 5. Conclusion In the present study, two isolates from the rhizomes of I. kashmiriana identified as isonigricinc (1) and isoirisolidone (2) have been evaluated for their immunodulating activity. Isoflavone 1 showed a dose dependent suppressive effect on CD4+ and CD8+ T-cells and cytokines under study and thereby having an immunosuppressive activity. Compound 2 showed a dose dependent potentiating effect on CD4+, CD8+ T-cells and cytokines thereby showing an immunostimulatory effect. The methylated derivatives of compound 1 and 2 in flowcytometric studies showed a stimulatory effect on interleukins under study but had suppressive effect on T-cells and IFN-␥ production. The activity can be correlated to the position and nature of substitution on the isoflavone backbone. The study suggests the potential of isoflavones 1 and 2 as prospective immunomodulatory agents. Disclosure of interest The author declares that he has no conflicts of interest concerning this article. Acknowledgments The author expresses his gratitude to Dr. Sarang Bani and Dr. Sheikh Fayaz Ahmad, Pharmacology Division, Indian Institute of Integrative Medicine (IIIM), Jammu, for providing me the facilities of performing the experiments with animals and carrying out the flowcytometric experiments. I also express my gratitude to my supervisors Dr. S. Koul, IIIM, Jammu, and Prof. M.A. Qurishi, Department of Chemistry, University of Kashmir for helpful suggestions. References [1] Iain BM, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol 2007;7:429–44. [2] Lubberts E. The role of IL-17 and family members in the pathogenesis of arthritis. Curr Opin Invest Drugs 2003;4:572–7. [3] Atkinson MA, Eisenbarth GS. Type 1 diabetes: new perspectives on disease pathogenesis and treatment. Lancet 2001;358(9277):221–9. [4] Van der Fits L, van der Wel LI, Laman JD, Prens EP, Verschuren MC. Psoriatic lesional skin exhibits an aberrant expression pattern of interferon regulatory factor-2 (IRF-2). J Pathol 2003;199:107–14. [5] Powrie F, Coffman RL. Cytokine regulation of T-cell function: potential for therapeutic intervention. Immunol Today 1993;14:270–4. [6] Liblau RS, Singer SM, McDevitt HO. Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol Today 1995;16:34–8. [7] Mastan SK, Saraseeruha A, Gourishankar V, Chaitanya G, Raghunandan N, Reddy AG, et al. Immunomodulatory activity of methanolic extract of Syzygiumcumini seeds. Pharmacol Online 2008;3:895–903. [8] Distad BJ, Amato AA, Weiss MD. Inflammatory myopathies. Curr Treat Opt Neurol 2011;13(2):119–30. [9] Niesvizky R, Naib T, Christos PJ, Jayabalan D, Furst JR, Jalbrzikowski J, et al. Lenalidomide-induced myelosuppression is associated with renal dysfunction: adverse events evaluation of treatment-naive patients undergoing frontline lenalidomide and dexamethasone therapy. Br J Haematol 2007;138:640–3. [10] Raza A, Meyer P, Dutt D, Zorat F, Lisak L, Nascimben F, et al. Thalidomide produces transfusion independence in long-standing refractory anemias of patients with myelodysplastic syndromes. Blood 2001;98:958–65. [11] Middleton Jr E, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 2000;52:673–751.
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Original article
Immunomodulatory activity of isoflavones isolated from Iris kashmiriana: Effect on T-lymphocyte proliferation and cytokine production in Balb/c mice Nighat Nazir Department of Chemistry, Islamia College of Science and Commerce, Hawal, Srinagar 190002, J&K, India
a r t i c l e
i n f o
Article history: Received 9 November 2012 Accepted 12 December 2012 Keywords: Iris kashmiriana Iridaceae Isoflavones Cytokines Immunomodulators Flowcytometry
a b s t r a c t This study deals with the evaluation of immunomodulatory action of a new isoflavone isonigricin (1) and first report isoirisolidone (2) isolated from rhizomes of Iris kashmiriana. Flowcytometric method was employed to study their effect on production of T-lymphocytes (CD4+ and CD8+ T-cells) and T-cell cytokines (IL-2, IL-4 and IFN-␥) in a dose-dependant manner. Drugs at doses of 0.025 to 0.8 mg/kg per oral dose showed compound 1 to possess immunosuppressant activity on T-cells as well as cytokines, while compound 2 acted as immunostimulator for both cells and cytokines. However, their methylated products viz. 1OMe and 2OMe exhibited stimulatory effect on interleukins under study and suppressive effect on production of T-lymphocytes and IFN-␥. The oral LD50 in mice was observed to be more than 200 mg/kg body weight. Such a difference in their activity is the manifestation of the position and nature of substituents present on isoflavonoidal skeleton and demonstrates the potential of these isolates as prospective immunomodulatory agents. © 2013 Published by Elsevier Masson SAS.
1. Introduction The immune system is a highly complex and extraordinarily sophisticated network involving proteins, special cells, tissues and organs that work together to defend the body against invasion by foreign individuals like germs, parasites, toxins etc. Thus, it acts as the body’s defence against infections that works through a series of steps called the immune response. Prominent immuno factors of antigen-specific immune responses are T-lymphocytes (including T-helper cells, T-suppressor cells and cytotoxic T-cells) and their cytokines. The healthy state is believed to be based on a fine-tuning of various humoral and cellular factors functioning in the immunoregulatory mechanism. Any sort of imbalance in the production or expression of these immune factors can result in a pathological condition. A number of factors like food, drugs, pollutants, physical and psychological stress, hormones etc. can influence this balance, resulting in either immunostimulation or immunosuppression. Such conditions have been shown to be involved in the etiology as well as pathophysiology of many diseases of skin, gut, respiratory tract, joints and central organs [1–4]. The role of cytokines in the pathogenesis of autoimmune diseases has been the subject of intense study in recent years. For example, type-1 cytokines (IL-2, IFN-␥, TNF-␣) are involved in autoimmune diseases such as type-1 diabetes, multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases and acute graft versus host
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disease. On the contrary, type-2 cytokines (like IL-4, IL-5, IL-12) are associated in allergic response and inhibit cell-mediated immunity [5,6]. Modulation of immune response through stimulation or suppression may help in maintaining a disease-free state [7]. Modification of immune function by pharmacological agents is emerging as a major area of therapeutics. In recent years, there has been great interest in the pharmacological manipulation of the immune response. Selective pharmacological action on an individual component of a complex immune response is seen as a particularly attractive approach to the therapy of immunologically mediated diseases. Immunomodulatory activity is a collective term indicating biological or pharmacological effect on various factors functioning in the immune response which requires timely interaction of multiple cell types and its modulation aims to restore immune homeostasis. Immunomodulators are the agents that help in restoring a proper balance of these immune factors and are effective for treating and preventing diseases and illnesses that stem from such imbalances. Immunomodulators can be either stimulators or suppressors. Former are helpful in preventing immunodeficiencies or for generalized immunosuppression following drug treatment, for combination therapy with antibiotics, and as adjuvants for vaccines and other depressed states of immunity. Those metabolites that suppress immune reactions are potentially useful to mitigate autoimmune diseases such as systemic lupus erythmatosus, hypersensitivity reactions, graft rejection or certain gastrointestinal tract diseases. Presently available immunomodulatory drug have side effects such as hypertension, renal failure, oncogenicity, hypertrichosis [8], myelosuppression [9], anemia [10] and are
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expensive [8]. Moreover, constant increase in antibiotic resistant strains of microorganisms has prompted scientists to look for herbal immunomodulators to treat various infections. Modulation of immune functions using medicinal plants and their products as a possible therapeutic measure has become a fundamental principle of therapeutic approach. Flavonoids are prominent plant secondary metabolites with variable phenolic structures and are consumed by humans as dietary constituents. Although not considered nutrients and essential for life, flavonoid ingestion may play a significant role in health and disease [11,12]. Isoflavones is a large subclass of flavonoids with 15-carbon (C6-C3-C6) backbone. Numerous studies have shown an association between isoflavone rich dietary consumption and reduced cancer risk, particularly breast and prostrate cancer [13–15]. It has been shown that some isoflavones can act as inhibitors of multidrug resistance transporter MRP1 through influencing the biophysical properties of membranes [16]. The preventive role of isoflavones in cancer, cardiovascular diseases, osteoporosis, obesity and menopausal symptoms [13,17–19], in addition to their antioxidant [20,21], antimicrobial [22], antiinflammatory and estrogenic activities [14,15] has been largely documented. The estrogenic activity of genistein, daidzen and equol are currently being extensively investigated at the molecular, pre-clinical and clinical levels to determine their potential for treatment of chronic diseases such as hormone-dependent cancer, cardiovascular diseases and osteoporosis [23]. Flavonoids have also been reported to show immunomodulatory properties by affecting varied cells of immune system. Recent reports [24,25] indicate that several types of flavonoids stimulate human peripheral blood leukocyte proliferation. They significantly increase the activity of helper T-cells, cytokines, interleukin 2 (IL-2), interferon ␥ (IFN-␥) and macrophages and are thereby useful in the treatment of several diseases caused by immune system dysfunction. Luteolin, 3 ,4 ,5,7tetrahydroxyflavone, an important member of the flavonoid family has shown to exert immunomodulatory effects that may be beneficial in the treatment of neurodegenerative diseases such as multiple sclerosis, which has an underlying T-cell mediated autoimmune pathology [24,25]. We have previously reported the immunomodulatory effect of two isoflavones, irilone and irisolidone on the production of T-lymphocytes (CD4+ and CD8+) and T-cell cytokines namely IL-2, IFN-␥, IL-4 and IL-5 that was found to be dependent on the position and nature of substitution [26]. However, methylation of these compounds drastically decreased their activities. The wide spectrum of pharmacological activities associated with the isoflavones and the growing interest in investigating natural isolates for possible T-cell modulating properties, prompted us to undertake the chemical investigation of Iris kashmiriana (reported to be a rich source of isoflavones) [27,28] and then screen the pure isolates for their immunomodulatory activity. Therefore, the present report deals with the evaluation of immunomodulatory activity of new isoflavone isonigricin (1) and a first report isoirisolidone (2) isolated previously [28] from the rhizomes of I. kashmiriana and their O-methylated products (1OMe and 2OMe) with respect to T-lymphocytes (CD4+ and CD8+ cells) and T-cell cytokine (IL-2, IL-4 and IFN-␥) production in mice using flowcytometry. The results presented merely point to the effect of the isoflavones under study on the production of T-lymphocytes and cytokines and hence to their potential prospective clinical application as immunomodulatory agents. 2. Materials and methods 2.1. General Melting points were determined in one end open capillaries on Buchi 570 melting point apparatus and are uncorrected. IR spectra
were determined in KBr pellets on Perkin–Elmer spectrometer. 1 H and 13 C-NMR were recorded on a Bruker 200 MHz spectrometer using TMS as internal standard. Mass spectra were determined on Jeol-MSD300 and Bruker Esquire 3000. Column chromatography was carried out on Silica-gel (60–120 mesh size, E. Merck). TLCs were run on 0.25 mm silica gel 60 F254 plates using UV light or Ceric ammonium sulfate solution for detection of the spots. Fluorisothiocyanate (FITC)–labeled CD4 anti-mouse monoclonal antibody, Phycoerythrin (PE)–labeled CD8 anti-mouse monoclonal antibody, FACS lysin solution (BD Bioscience, USA), Levamisole, cyclosporinA (Sigma-Aldrich, India), wash buffer, mouse cytokine standards (IL-2, IFN-␥, IL-4, IL-5) (BD Biosciences, USA) were used. Unless and otherwise specified, all the solvents were of analytical grade (Qualigens). 2.2. Plant material The rhizomes of I. kashmiriana were purchased from Nehru Botanical Garden, Srinagar (J&K, India), identified at Institute of Plant Taxonomy, University of Kashmir, under Acc. No. 13595 and Collection No. 1207 GH DAR. 2.3. Extraction and isolation Air dried and powdered rhizomes of the plant (1.5 kg) were extracted with Petroleum ether (4 l, 60–80 ◦ C, Soxhlet, 48 hours) and the extract concentrated and reduced to 250 ml volume and left overnight at room temperature (20–22 ◦ C) when a pale yellow solid precipitated out and was filtered. The crude material was dissolved in a mixture of chloroform:hexane (7:3) and left at 0 ◦ C to give a crystalline solid which on filtration and recrystallisation in the same solvent system afforded fine needles of compound 2 (138 mg, 0.0092% yield, mp 220 ◦ C). The defatted rhizomes were extracted with methanol (4 l, soxhlet, 36 hours) and the extract de-solventised and lyophilized (using Christ Alpha 1-4 lyophiliser) to give dried material (68 g). Chromatography of the crude extract (60 g) was carried out on silica gel (60–120 mesh) using an increasing gradient of n-hexane:chloroform followed by increasing gradient of chloroform:methanol mixture. Fractions of 200 ml volume each were collected, analyzed by TLC and pooled accordingly. Fractions 25-30 eluted with 1:3n-hexane:chloroform, pooled, concentrated and rechromatographed in n-hexane: chloroform yielded 93 mg of 2. Fractions 57-61 (95:5 chloroform:methanol) were combined and concentrated to yield a pale yellow amorphous powder (430 mg) which on dissolution in methanol (50 ml, reflux conditions) followed by cooling afforded colorless shiny crystals of 1 (355 mg, 0.023% yield, mp 249 ◦ C). 2.4. Preparation of methyl derivatives of compound 1 and 2 (1OMe and 2OMe) To a methanolic solution of compound 1 (100 mg, 0.32 mmol, 50 ml), was added an ethereal solution of diazomethane (50 ml) (prepared from 5 g of nitrosomethyl urea) and the contents were left at 10 ◦ C for 8 hrs. The contents were concentrated and the residue column chromatographed on silica gel (60–120 mesh size) and eluted with 10% ethyl acetate in n-hexane to give 1OMe – a white solid (76 mg, 0.23 mmol, 71.8% yield). The compound 2OMe was prepared from compound 2 (80 mg, 0.25 mmol) by the procedure as described above (63 mg, 0.184 mmol, 73.6% yield). 2.5. Spectral data 2.5.1. Spectral data of compound 1 Colorless solid analysed for C17 H12 O6 (Cal. C 65.38 H 3.873% found C 65.98 H 3.941%); mp 249 ◦ C; UV (MeOH): Max 213, 264
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& 322 nm; IR (KBr): max = 3267, 2952, 2914, 1632, 1591, 1516, 1499, 1467, 1439, 1371, 1343, 1287, 1259, 1215, 1172, 1128, 1104, 1079, 1057, 1036, 946, 926, 847, 835, 823, 755, 714, 666 cm−1 ; 1 HNMR (200 MHz, DMSO-d6 )␦(ppm) = 3.92 (3H, s, OMe), 6.12 (2H, s, -OCH2 O-), 6.81 (2H, d, J = 8.43 Hz, 3 ,5 -H), 7.02 (1H, s, 5-H), 7.36 (2H, d, J = 8.41 Hz, 2 , 6 -H), 8.20 (1H, s, -OCH C); 13 C-NMR (125 MHz, DMSO-d6 )␦(ppm) = 152.53, 122.37 174.09, 103.54, 135.86, 153.93, 140.41, 151.01, 1133.16, 102.58, 124.20, 130.22, 114.83, 157.06, 114.83, 130.22, 60.77; MS: m/z (%): 312 (100), 297 (100), 244 (50), 219 (23), 194 (36), 152 (37), 146 (12), 118 (7) and 93 (9). 2.5.2. Spectral data of compound 1OMe A white solid; mp 144–45 ◦ C; IR (KBr): max = 2964, 2920, 1644, 1605, 1508, 1473, 1432, 1393, 1356, 1308, 1285, 1257, 1241, 1215, 1175, 1099, 1077, 1053, 1035, 945, 925, 913, 852 cm−1 ; 1 H-NMR (500 MHz, CDCl3 )␦(ppm) = 3.84 (3H, s, OMe), 4.08 (3H, s, OMe), 6.07 (2H, s, -OCH2 O-), 6.63 (1H, s, 5-H), 6.93 (2H, d, J = 8.6 Hz, 3 ,5 -H), 7.12 (2H, d, J = 8.6 Hz, 2 ,6 -H), 7.68 (1H, s, -OCH C); 13 CNMR (50 MHz, CDCl3 )␦(ppm) = 55.32, 61.25, 93.24, 102.16, 113.82, 113.88, 124.13, 125.33, 130.37, 135.57, 135.81, 140.70, 150.23, 152.78, 154.72, 154.80, 159.51, 175.46; MS: m/z (%): 326 (84) 298 (77), 297 (100), 280 (74), 252 (49), 194 (34), 164 (42), 151 (29), 132 (88), 93 (23), 89 (51). 2.5.3. Spectral data of compound 2 A pale yellow crystalline solid analyzed for C17 H14 O6 (Cal. C 64.97 H 4.89% found C 65.71 H 4. 52%); mp 220 ◦ C; UV (MeOH): Max 213, 265 & 340 nm (+AlCl3 : 237, 269, 312, 364 nm, +AlCl3 + HCl 233, 274, 312, 362 nm, NaOAc 222, 273, 341 nm, NaOAc + H3 BO3 221, 267 nm); IR (KBr): max = 3438, 3068, 2989, 2959, 1660, 1624 cm−1 ; 1 H-NMR (500 MHz, CDCl )␦(ppm) = 3.85 & 4.04 (3H each, s, 2x3 OMe), 6.53 (1H, s, 8-H), 6.99 (2H, dd, J = 2.0 & 8.74 Hz, -3 ,5 -H), 7.46 (2H, dd, J = 2.0 & 8.71 Hz, 2 ,6 -H), 7.88 (1H, s, -OCH C); 13 C-NMR (125 MHz, CDCl3 )␦(ppm) = 155.16, 122.89, 181.28, 152.64, 130.37, 153.47, 93.21, 152.83, 106.46, 123.12, 130.15, 114.12, 159.80, 114.12, 130.15, 55.37, 56.89; MS: m/z (%): 314 (100), 299 (50), 252 (7), 207 (52), 203 (11), 192 (6) & 164 (12). 2.5.4. Spectral data of compound 2OMe A white solid; mp180 ◦ C; IR (KBr): max = 2947, 2923, 1642, 1606, cm−1 ; 1 H-NMR (200 MHz, CDCl3 )␦(ppm) = 3.88, 3.96, 3.98 & 4.01 (12H, s, 4x-OMe), 6.65 (1H, s, 8-H), 6.98 (2H, d, J = 8.8 Hz, 3 ,5 -H), 7.43 (2H, d, J = 8.8 Hz, 2 ,6 -H); 13 C-NMR (50 MHz, CDCl3 )␦(ppm) = 53.30, 56.20, 61.50, 62.00, 94.01, 105.72, 114.18, 122.73, 123.04, 130.13, 129.86, 152.31, 153.24, 154.56, 155.75, 159.70, 175.30; MS: m/z (%): 342 (34), 327 (88), 312 (100), 299 (11), 195 (17), 182 (9), 154 (14), 132 (15), 124 (17), 122 (29). 2.6. Animals and drug Eight to 10-week-old Balb/C mice (Mus musculus), 18–22 g body weight in groups of six were employed for the study. The animals were housed under standard conditions of temperature (23 ± 2 ◦ C), relative humidity (55 ± 10%), 12 hours photoperiod and fed with standard pellet diet (Lipton India Ltd) and water ad libitum. In every experiment, one group of animals was used as a sensitized vehicle control and another as a non-sensitized naïve control. Drugs were freshly prepared as a homogenized suspension in 1% w/v acacia gum and administered orally daily once a day for the duration of experiment. Cyclosporin (immunosupressant) and Levamisole (immunostimulant) were used as standard drugs and were administered once daily at 5 mg/kg and 2.5 mg/kg per oral dose respectively. The treated groups received drugs at 0.025, 0.05, 0.10, 0.20, 0.40 and 0.80 mg/kg per oral dose.
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2.7. Antigen One hundred microliter of ovalbumin (2 mg/ml PBS) was injected intraperitonally in experimental mice for immunization. Drugs were administered for seven days including the day of immunization. On eighth day, animals were challenged by injecting same amount of ovalbumin i.p to induce the challenge. 2.8. Acute Toxicity Test Acute Oral Toxicity studies were carried out following OECD Guidelines no. 423. After a single dose treatment with drug extract (200 mg/kg per oral dose) to respective groups (female mice only), the animals were observed continuously for 1 hour and then half hourly for 5 hours for any change in general behavior and finally up to 15 days for any mortality. 2.9. Study design for CD4+ and CD8+ T cell estimation One hundred microliter of whole blood, with 10 l of Mab (monoclonal antibody) FITC (fluorescein isothiocyanate) anti-CD4 and 10 l of Mab (monoclonal antibody) PE (Phycoerythrin) antiCD8 were added and mixed gently. Tubes were incubated in dark for 30 minutes at room temperature. Samples were again incubated at room temperature for 10 minutes after adding 2 ml of 1X FACS lyses solution (BD Biosciences). Samples were spinned (300–400Xg) for 10 minutes at room temperature. The supernatant was aspirated and sample was given three washings of phosphate buffer saline (pH). The resulting stained cell pellet was resuspended in 500 l of phosphate buffer saline and was run on a flow cytometer (LSR, BD Biosciences). Acquisition and the analysis were done using Cell Quest Pro software (BD Biosciences). 2.10. Study design for IL-2, IFN- (Th1) and IL-4 (Th2) cytokine estimation To detect the intracellular expression of cytokines, anti-CD4FITC, anti-interleukin (IL-2) PE, anti-interleukin (IL-4) PE, and anti-CD8 PE, anti-interferon (IFN-␥) FITC was analysed. A total of 100 l of whole blood was incubated with 10 l of Mab (monoclonal antibody) FITC-anti-CD4 in the dark incubation for 30 min at room temperature. Erythrocytes were lysed for 10 min incubation at room temperature with 2 ml 1X FACS Lysing solution (Becton Dickinson) and centrifugation at 300Xg for 10 min. After the cells were washed once with 1 ml phosphate buffer solution (PBS)/0.5% BSA by centrifugation at 300Xg for 5 min. Supernatants were removed and 500 l of permeabilization solution and 10 l of each monoclonal antibody, anti-IFN-␥ FITC, anti-IL-2 PE and anti IL-4 PE was added to separate the tubes containing the cell pellets. The samples were votexed and incubated for another 30 min at room temperature. The permeabilized cells were finally washed in 1 ml PBS/0.5% BSA by centrifugation at 300Xg for 5 minutes. Supernatants were removed and the cells were resuspended in 500 l 1% PFA, and stored at 2–8 ◦ C in the dark. The fixed cells were then analyzed within 24 h in a flow cytometric analyzer using Cell Quest Pro software (BD Biosciences). 2.11. Statistical analysis The significance of differences between control and treated groups was analyzed using Student’s t-test. Differences were considered significant at P ≤ 0.01 and significant values are represented by asterisk marks. All experiments were repeated at least three times. Data are expressed as % mean ± SE.
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50
OMe O
O
CD4+
(a)
1 1OMe 2 2OMe
40 **
O
**
O
O
MeO
(b)
**
(%) Mean±SE
OH
30
*
OH
**
** **
20
**
**
** ** ** ** ** ** **
**
**
O
**
**
HO
* **
*
*
**
10
OMe
Compounds 1 and 2 were identified as isonigricin and isoirisolidone respectively (Fig. 1) on the basis of their physical and spectral analyses like IR, NMR and mass spectrometry as previously reported by us [28].
0.8 mg/kg
0.400 mg/kg
0.200 mg/kg
0.100 mg/kg
0.050 mg/kg
0.025 mg/kg
Cyclosporine 5mg/kg
3.1. Identification of isoflavones
Cont. (Sensitised)
Cont. (Naïve)
0
3. Results
Levamisole 2.5mg/kg
Fig. 1. Structure of compound (a) 1 and (b) 2.
Fig. 2. Percentage age change in CD4+ cell expression in Balb/c mice treated with different concentrations of 1, 1OMe, 2 and 2OMe. Results are expressed as mean ± SE for group of six mice, and are representative of these experiments. Statistical significance, * P < 0.01, ** P < 0.001 when compared to control (sensitized) mice.
3.2. Effect on general behavior and acute toxicity
30
CD8+
1 1OMe 2 2OMe
**
20
**
3.4. Effect on Th1 cytokines (IFN- and IL-2) and Th2 cytokine (IL-4) Figs. 4–6 show dose dependent effect of 1, 2, 1OMe and 2OMe at doses 0.025 to 0.800 mg/kg per oral dose, on IL-2, IFN-␥ and IL-4 cytokine expression respectively. The results show significant suppressive effect of 1 (close to suppressive effect of standard drug cyclosporin), being maximum at 0.40 mg/kg dose level, while
** ** **
** **
* *
**
**
** ** **
0.400 mg/kg
0.100 mg/kg
0.050 mg/kg
**
0.025 mg/kg
Cyclosporine 5mg/kg
Levamisole 2.5mg/kg
Cont. (Sensitised)
0
**
** **
**
**
**
** *
10
Cont. (Naïve)
Dose dependent effect of isoflavone1, 2, 1OMe and 2OMe at doses 0.025 to 0.800 mg/kg per oral dose, on CD4+ and CD8+ T-cells using flow cytometry are presented in Figs. 2 and 3 respectively. Animals treated with isoflavone1 and 2 showed suppression and stimulation respectively in CD4+ and CD8+ T-cells expression in comparison with sensitized control. The maximum suppressive effect of 1 was observed at a dose level of 0.80 mg/kg where it was 20.38% while maximum stimulatory effect of 21.93% for 2 was observed at a dose level of 0.05 mg/kg being even more than standard drug Levamisole (18.01%) at 2.5 mg/kg dose level in case of CD8+ T-cells. Methylated product of 1 (1OMe) again showed suppressive effect on CD4+ T-cell expression but for CD8+ T-cell expression, it showed stimulatory effect with maximum activity at dose level of 0.025 mg/kg. On the other hand, 2OMe exhibited suppress give effect on CD4+ and CD8+ cells contrary to 2. The maximum suppression by 2OMe was at dose level of 0.025 mg/kg that was comparable to immunosuppressive standard drug cyclosporin at dose level of 2.5 mg/kg.
(%) Mean±SE
**
0.200 mg/kg
3.3. Effect on CD4+ and CD8+ T cells
0.8 mg/kg
A single dose of drugs administered in each group of mice did not show any change in general behavior of the test animals. No mortality was observed over a period of 15 days up to the dose level of 200 mg/kg orally. So acute LD50 was found to be greater than 200 mg/kg.
Fig. 3. Percentage age change in CD8+ cell expression in Balb/c mice treated with different concentrations of 1, 1OMe, 2 and 2OMe. Results are expressed as mean ± SE for group of six mice, and are representative of these experiments. Statistical significance, * P < 0.01, ** P < 0.001 when compared to control (sensitized) mice.
stimulatory effect of 2 (more than standard drug Levamisole for IL-4 cytokine expression), being maximum at 0.05 mg/kg of dose level. 1OMe, contrary to 1, showed stimulatory effect on IL-2 and IL-4 cytokine expression with maximum effect at 0.05 mg/kg of dose level while suppressive effect on IFN-␥ being maximum at
N. Nazir / Biomedicine & Preventive Nutrition 3 (2013) 151–157
IL-2
40
1 1OMe 2 2OMe
**
20
155
IL-4
1 1OMe 2 2OMe
30 ** **
*
*
****
**
**
** ** **
*
**
10
**
**
**
** **
** **
Levamisole 2.5mg/kg
Fig. 6. Percentage age change in IL-4 expression in Balb/c mice treated with different concentrations of 1, 1OMe, 2 and 2OMe. Results are expressed as mean ± SE for group of six mice, and are representative of these experiments. Statistical significance, * P < 0.01, ** P < 0.001 when compared to control (sensitized) mice.
4. Discussion
50
IFN-γ
1 1OMe 2 2OMe
40 ** **
(%) Mean±SE
Cont. (Sensitised)
Cont. (Naïve)
0.8 mg/kg
0.400 mg/kg
0.200 mg/kg
0.100 mg/kg
0.050 mg/kg
0.025 mg/kg
Cyclosporine 5mg/kg
Levamisole 2.5mg/kg
Cont. (Sensitised)
Cont. (Naïve)
Fig. 4. Percentage age change in IL-2 expression in Balb/c mice treated with different concentrations of 1, 1OMe, 2 and 2OMe. Results are expressed as mean ± SE for group of six mice, and are representative of these experiments. Statistical significance, * P < 0.01, ** P < 0.001 when compared to control (sensitized) mice.
**
**
**
0
Cyclosporine 5mg/kg
0
**
** **
**
**
**
**
0.8 mg/kg
** **
**
** **
*
**
0.050 mg/kg
**
**
**
** **
0.400 mg/kg
**
0.025 mg/kg
**
10
**
20
0.200 mg/kg
**
**
0.100 mg/kg
**
**
(%) Mean±SE
(%) Mean±SE
**
30 ** **
**
**
**
20
** **
10
** **
** ** **
**
** **
** **
**
**
**
** **
** **
0.8 mg/kg
0.400 mg/kg
0.200 mg/kg
0.100 mg/kg
0.050 mg/kg
0.025 mg/kg
Cyclosporine 5mg/kg
Levamisole 2.5mg/kg
Cont. (Sensitised)
Cont. (Naïve)
0
Fig. 5. Percentage age change in IFN-␥ expression in Balb/c mice treated with different concentrations of 1, 1OMe, 2 and 2OMe. Results are expressed as mean ± SE for group of six mice, and are representative of these experiments. Statistical significance, * P < 0.01, ** P < 0.001 when compared to control (sensitized) mice.
0.80 mg/kg of dose level. 2OMe also showed stimulation of IL-2 and IL-4 cytokine expression at higher dose levels in tune with 2 but a suppressive effect on IFN-␥ with maximum effect at 0.025 mg/kg dose level.
Modulation of immune response through stimulation or suppression may help in maintaining a disease-free state. Immunopharmacology is a comparatively new and developing branch of pharmacology aiming at searching immunomodulating agents with no or not very serious side effects. There is, thus, a growing interest in identifying and characterizing natural compounds with immunomodulatory activity. Our results show the immunomodulating activity of isoflavones 1 and 2, indicating their possible role in treating pathophysiological conditions involving immunological background. The compound 1 induced a doserelated inhibition of CD4+ and CD8+ T-cell expression (Figs. 2 and 3) and cytokine IL-2, IFN-␥ and IL-4 production (Figs. 4–6). T-cells expressing CD4 are increased when there is a general expansion due to increased immunological activity of T-cells. Thus an inhibition of this expression by 1 in a dose-dependent manner is an indication of its immunosuppressive activity. Due to its effect on T-cells and B-cells, IL-2 is a central regulator of immune responses. Under physiological conditions, IL-2 is produced mainly by T-cells expressing the surface antigen CD4 following cell activation by mitogens or allogens. Thus suppression of CD4+ T-cells inhibits IL-2 presence and is also responsible for inhibition of IL-4 production by CD4+ T-cells. Since IL-2 functions to stimulate the synthesis of IFN-␥ by T-lymphocytes and natural killer (NK) cells and also stimulates the differentiation of naïve CD4 T-cells into IFN-␥ producing Th-1 cells, the inhibition of IL-2 is possibly responsible for reduced IFN-␥ secretion by Th-1 type CD4+ and CD8+ cells. These findings demonstrate that 1 has a potent T-cell and cytokine suppressive activity and suggestive of its possible therapeutic usefulness, especially to mitigate autoimmune or certain gastrointestinal tract diseases and most importantly against allograft rejection which has been shown to be associated with an increase in IL-2 and other pro-inflammatory cytokines such as IFN␥ and TNF-␣. The most effective transplantation immunosupressive strategies are based on interruption of IL-2 signalling by cyclosporin
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A and Tacrolimus. However, treatment with these drugs is associated with serious adverse side effects including nephrotoxic, diabetogenic, neurological and cardiovascular effect [29]. Apparent safety over long-term administration of 1 is encouraging enough to warrant further studies to explore its possible therapeutic role in modern clinical practice. Dose dependent potentiating effect of 2 on CD4+ and CD8+ Tcells (Figs. 2 and 3) along with the increase of IL-2, IFN-␥ and IL-4 (Figs. 4–6) secretion shows its immunostimulatory effect, being even more than standard drug levamisole in case of CD8+ T-cells. The LD50 of 2 has been shown to be greater than 200 mg/kg body weight, its stimulatory effect at a dose level as small as 0.800 mg/kg body weight in comparison to the dose level of 2.5 mg/kg body weight of levamisole suggests it as a potential immunostimulant. Moreover, adverse side effects have been attributed to levamisole. These are agranulocytosis – leaving patients susceptible to fulminate and opportunistic infections and can also cause debilitating cutaneous necrotizing vasculitis [30,31], multifocal inflammatory leukoencephalopathy [32,33] and it could be a triggering factor in multiple sclerosis [34]. This suggests the possible usefulness of 2 as immunomodulatory agent for treatment of cancer, immunodeficiency diseases, or for general immunosupression following drug treatment, for combination therapy with antibodies and as adjuvants for vaccines. The immunosuppressive effect 1 and immunostimulatory effect of 2 could be apparently attributed to the nature of the functional groups present in the two compounds as the basic nucleus in both the compounds is same i.e. an isoflavone nucleus. In order to explore the possibility that the functional groups in ring A and B of 1 and 2 could be responsible for the said effect, an examination of the structure of 1 and 2 shows a free phenolic group at 4 position in ring B of 1 which is present as OMe group in compound 2. In ring A of compound 1, no free phenolic groups is present, which however is present in compound 2 showing thereby higher pka in ring A of 1 compared to that of ring A of 2. Interestingly, three positions in ring A of both the compounds are substituted. Two possibilities could be comptemplated namely i) the substituents in the ring A or ii) 4 substituent in ring B responsible for the antagonistic effect. We, therefore, decided to methylate compounds 1 and 2 and subject them to flow cytometric studies and study the effect of decrease of the charges in the ring A and ring B. The results obtained showed that the methylation of 4 in ring B of compound 1 again had a suppressive effect on the CD4+, CD8+ T-cells and IFN␥ (Figs. 2, 3 and 5) but had a stimulating effect on IL-2 and IL-4 which increases with an increase in dose level. Thus, it is in contrary to its 4’-demethylated product 1 (Figs. 4 and 6). In case of compound 2, there was significant change in activity after methylation. A decrease in the stimulatory effect could be seen for IL-2 and IL-4 while as the values of CD4+ and CD8+ T-cells and IFN␥ shifted towards suppression at several dose levels. Figs. 2 to 6 show the %age increase/decrease of the immunomodulating activities of the parent compounds and their methylated derivatives. The results are suggesting that free hydroxyls in ring A of isoflavones can be responsible for overall stimulatory activity of 2 whereas free hydroxyls in ring B can cause overall suppressive activity of 1. Switching over of suppressive effect to stimulation for IL-2 and IL-4 by methylation of 4 hydroxyl group of 1 in tune with stimulatory effect of 2 and 2OMe (both having methylated 4 position) for IL-2 and IL-4, suggest that expression of these two immune factors is favoured by methylated substitution in ring B. In the same manner changing of stimulatory effect of 2 for IFN-␥, CD4+ and CD8+ T-cells by methylation of ring A hydroxyls to suppressive effect, that is also observed with 1 and 1OMe suggests that free hydroxyl substitution in ring A could be a contributing factor for observed enhanced stimulation of these immunofactors. Thus, it could be concluded that the nature of substituents in ring A or in
other terms the pKa in ring A may be responsible for the observed suppressant or stimulation effect. It may be said that, there could be other factors like the position of the functional groups and their nature in the ring A which may also be responsible for the said effect. These findings are in quite agreement with the earlier studies where the importance of position and number of phenolic substitution on isoflavone backbone for immunomodulatory activity has been discussed [26,35]. 5. Conclusion In the present study, two isolates from the rhizomes of I. kashmiriana identified as isonigricinc (1) and isoirisolidone (2) have been evaluated for their immunodulating activity. Isoflavone 1 showed a dose dependent suppressive effect on CD4+ and CD8+ T-cells and cytokines under study and thereby having an immunosuppressive activity. Compound 2 showed a dose dependent potentiating effect on CD4+, CD8+ T-cells and cytokines thereby showing an immunostimulatory effect. The methylated derivatives of compound 1 and 2 in flowcytometric studies showed a stimulatory effect on interleukins under study but had suppressive effect on T-cells and IFN-␥ production. The activity can be correlated to the position and nature of substitution on the isoflavone backbone. The study suggests the potential of isoflavones 1 and 2 as prospective immunomodulatory agents. Disclosure of interest The author declares that he has no conflicts of interest concerning this article. Acknowledgments The author expresses his gratitude to Dr. Sarang Bani and Dr. Sheikh Fayaz Ahmad, Pharmacology Division, Indian Institute of Integrative Medicine (IIIM), Jammu, for providing me the facilities of performing the experiments with animals and carrying out the flowcytometric experiments. I also express my gratitude to my supervisors Dr. S. Koul, IIIM, Jammu, and Prof. M.A. Qurishi, Department of Chemistry, University of Kashmir for helpful suggestions. References [1] Iain BM, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol 2007;7:429–44. [2] Lubberts E. The role of IL-17 and family members in the pathogenesis of arthritis. Curr Opin Invest Drugs 2003;4:572–7. [3] Atkinson MA, Eisenbarth GS. Type 1 diabetes: new perspectives on disease pathogenesis and treatment. Lancet 2001;358(9277):221–9. [4] Van der Fits L, van der Wel LI, Laman JD, Prens EP, Verschuren MC. Psoriatic lesional skin exhibits an aberrant expression pattern of interferon regulatory factor-2 (IRF-2). J Pathol 2003;199:107–14. [5] Powrie F, Coffman RL. Cytokine regulation of T-cell function: potential for therapeutic intervention. Immunol Today 1993;14:270–4. [6] Liblau RS, Singer SM, McDevitt HO. Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol Today 1995;16:34–8. [7] Mastan SK, Saraseeruha A, Gourishankar V, Chaitanya G, Raghunandan N, Reddy AG, et al. Immunomodulatory activity of methanolic extract of Syzygiumcumini seeds. Pharmacol Online 2008;3:895–903. [8] Distad BJ, Amato AA, Weiss MD. Inflammatory myopathies. Curr Treat Opt Neurol 2011;13(2):119–30. [9] Niesvizky R, Naib T, Christos PJ, Jayabalan D, Furst JR, Jalbrzikowski J, et al. Lenalidomide-induced myelosuppression is associated with renal dysfunction: adverse events evaluation of treatment-naive patients undergoing frontline lenalidomide and dexamethasone therapy. Br J Haematol 2007;138:640–3. [10] Raza A, Meyer P, Dutt D, Zorat F, Lisak L, Nascimben F, et al. Thalidomide produces transfusion independence in long-standing refractory anemias of patients with myelodysplastic syndromes. Blood 2001;98:958–65. [11] Middleton Jr E, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 2000;52:673–751.
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