Journal of Medicinal Plants Research Vol. 4(22), pp. 2340-2347, 18 November, 2010 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR10.459 ISSN 1996-0875 ©2010 Academic Journals

Full Length Research Paper

Immunosuppressive effects of B on human peripheral blood lymphocytes

Yaowalak U-pratya 1, Usaneeporn Lueangamornnara 1, Weena Jiratchariyakul 2 and Tanawan Kummalue 3*

1Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand. 2Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Thailand. 3Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.

Accepted 31 August, 2010

This study investigates the immunomodulating effects of cucurbitacin B, a Thai herbal plant; isolated from Trichosanthes cucumerima L., on PHA-stimulated human Peripheral Blood Mononuclear Cells (PBMCs). Stimulated PBMCs were cultured in the presence or absence of cucurbitacin B and then markers of cell activation were measured. The results showed that 0.5 and 0.25 µg/ml of cucurbitacin B significantly inhibited the expression of CD69+CD3+, and CD25+CD3+ whereas no significant inhibition from 0.05 µg/ml of cucurbitacin B was detected. The secretion of TNF-ααα from stimulated PBMC was significantly inhibited by cucurbitacin B. After 24 h of incubation, the total Cytotoxic Lymphocyte (CTL) activity was significantly reduced by 0.5 µg/ml of cucurbitacin B when compared to the control group (9.83 ± 0.95 and 40.63 ± 2.93, P = 0.0033) or compared with Phytohemagglutinin (PHA) stimulation (9.83 ± 0.95 and 70.09 ± 3.35, P < 0.0001). A similar pattern of CTL activity was found after 48 and 72 h of incubation. In conclusion, cucurbitacin B inhibited PBMC activation by PHA. This study provides evidences that cucurbitacin B could be used as immunosuppressive agent.

Key words: Cucurbitacin B, Phytohemagglutinin stimulated peripheral blood mononuclear cell, cytotoxic lymphocyte function, CD69.

INTRODUCTION

Trichosanthes cucumerina L., a plant known as “Buap- effects. The exception is T. cucumerina, which is Khom” in Thai, is in the family, commonly unedible because of its bitter taste inducing nausea and found in Southeast Asia and Australia. Plants in this symptoms (Sagium, 1997; Mcmakin et al., 1988). family are composed 110 genera and about 640 species. In other countries, such as India, this special group of The plants are mostly woody or herbaceous with climbing plants particularly their seeds and fruit, have been or trailing stems bearing tendrils and often arising from a prescribed to treat various diseases, such as infections tuberous rootstock. In Thailand, these plants grow wild and malignancies. Several investigations have along rivers in provinces such as Kanjanaburi. In Thai demonstrated the striking in vitro cytotoxic activities of traditional medicine, some of them have been used for plant isolated compounds, cucurbitacin B, against several their anti-helmintic, anti-diabetic, and anti-inflammatory human cancer cell lines such as breast and lung cancer cells (Jiratchariyakul et al., 1999; Kummalue et al., 2009). Several types of cucurbitacin compounds have been isolated from many plants (Yesilada et al., 1988; Peters *Correspondence author. E-mail: [email protected]. Tel: et al., 1999; Recio et al., 2004; Kummalue et al., 2009; 662-4181367. Fax: 662-4181367. Tien et al., 2010). These compounds are divided into twelve categories (Rungeler et al., 1999). Abbreviation: CUR, Cucurbitacin B; CTL, cytotoxic such as cucurbitacin B, D, E, I, dihydrocucurbitacin B, lymphocyte; PBMC, peripheral blood mononuclear cell; CFSE, cucurbitacin R have anti-inflammatory activity (Yesilada 5, 6-carboxyfluorescein diacetate succinimidyl ester. et al., 1988; Peters et al., 1999; Recio et al., 2004). U-pratya et al. 2341

Cucurbitacin R was found to inhibit the production of followed by centrifugation at 500 g for 5 min. Cells were washed in TNF-α from lymphocytes and macrophages (Escandell et PBS and fixed in 1% paraformaldehyde in PBS pH 7.0 and stored at 4°C until flow cytometric analysis was performed al., 2007). Cucurbitacin B is one of the most abundant (Pattanapanyasat et al., 1994). The 20,000 cells were analyzed forms of cucurbitacins (Peter et al., 1997; Frei et al., with FACSCalibur (Becton Dickinson, USA) using Cell Quest 1998). It has significant anti-inflammatory activity and is software. used traditionally to treat liver disease (Peter et al., 1997; Agil et al., 1999). Previous reports of cucurbitacin B ααα revealed that this compound significantly inhibited STAT3 TNF-  assay by ELISA which is one of the signal transducers and activator PBMC was cultured in a complete medium with 45 µg/ml PHA in transcription factors (Aggarwal et al., 2006). STAT3 is the absence or presence of 0.05, 0.25 and 0.5 µg/ml cucurbitacin B involved in the pathway of interleukin-6 (IL-6), an compared to unstimulated control for 24, 48 and 72 h. The cell culture supernatant was collected after incubation and TNF-α was important mediator of acute phase response in the R immune system (Mitsuyama et al., 2006; Nefedova et al., measured using ELISA kits (Quantikine ) from R and D system (USA), according to the manufacturer’s protocol. 2005). Therefore, the aim of this study is to further elucidate the effects of cucurbitacin B on the human immune response in terms of Peripheral Blood K562 target cell labeling with CFSE (K562*) Mononuclear Cells (PBMC) activation including cytolytic º activity response. K562 was cultured in a complete medium at 37 C, 5% CO 2. The cells were washed with PBS, adjusted to 4x10 6 cells/ml and stained with 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) MATERIALS AND METHODS (Molecular Probes, Eugene, OR, USA) at a final concentration of 1 µM in PBS (Lyons and Parish, 1994). The K562* (CFSE stained- Plant materials cell) was incubated at 37°C for 10 min with continuous shaking. At the end of the incubation period, the cells were washed three times º The green mature fruits were collected in October from with cold complete medium and maintained at 4 C until use. Nakornphathom Province, Thailand. The fruits were washed and pressed. The plant identification and the preservation of the The cytotoxic lymphocyte (CTL) function assays voucher specimen (BKF No. 70279) were carried out by Royal

Forest Department, Ministry of Natural Resources and Environment, The CTL function was performed using three colors Flow Bangkok, Thailand. Cytometric (FC) cytotoxic assay (Lyons and Parish, 1994; Marim et 6 al., 2003; Kasatori et al., 2005; Derby et al., 2001). 3x10 PBMCs

were treated with cucurbitacin B at a final concentration of 0.5 µg/ml Plant extraction for 24, 48 and 72 h in 12 well-plates (Corning, New York, USA). The normal control was cultured in the absence of stimulation whereas The fresh fruits (10 kg) were squeezed and yielded 800 mL juice, the positive control was stimulated with 45 µg/ml of PHA. After which was shaken with diethyl ether. The ethereal extract was left in incubation, the cells were washed with complete medium and 1x10 6 the fume hood, during the evaporation, the crystallization occurred. cells were co-cultured with 2x10 4 cells of K562* for 4 h at 37 ºC, 5% The crystalline mixture (22.4 mg) comprised cucurbitacin B as a CO 2. The apoptosis of K562* was then determined using Annexin main component with a trace of dihydrocucurbitacin B. V-PE Apoptosis Detection Kit I (BD, Biosciences, Pharmingen, USA). Shortly after, the cells were washed twice with cold PBS, resuspended with 1X binding buffer (provided by the KIT), stained Sample collection and lymphocyte culture with Annexin V-PE and 7-AAD 5 µl each, incubated at RT in darkness for 15 min and analyzed using FACSCalibur within one After informed consent, peripheral blood was collected from healthy hour. The minimum 100,000 cells were analyzed with FACSCalibur volunteers. PBMC was isolated by ficoll-hypaque centrifugation using Cell Quest software. The data was presented as the gradient. The cells were cultured in a Complete Medium (CM) percentage of the total cytolytic activity calculated using the composed of RPMI 1640 (Gibco BRL,USA) supplemented with 10% following formula: FBS (Cambrex, Bio Science, USA), 2 mM glutamine, 100 U of penicillin per ml and 100 µg of streptomycin per ml (all from Gibco The total cytolytic activity (%) = (% Annexin V+7AAD-) + (% º BRL,USA) and incubated in 5% CO 2, 37 C for 24 to 72 h. Annexin V+7AAD+) + (% Annexin V-7AAD+)

Stimulation and detection of activation markers on Statistical analysis lymphocytes The data were expressed as mean ± SD or mean ± SEM and analyzed by ANOVA using Stat VIEW 4.0 software. The statistical 1x10 6 cells/ml of PBMCs were cultured in a complete medium. The significance at level P < 0.05 was used for all comparisons. activation of lymphocytes was performed using a final concentration of 45 µg/ml of Phytohemagglutinin (PHA). Cucurbitacin B was added immediately at a final concentration of 0.05, 0.25, or 0.5 RESULTS µg/ml. After 24 h of incubation, the cells were harvested and stained with CD25-FITC, CD69-PerCP (both from BioLegend, USA) and CD3-APC (BD, Biosciences, Pharmingen, USA) antibodies for Activation maker expression 30 min at 4ºC in darkness. The cells were incubated with FACLyse (BD, Biosciences, Pharmingen, USA) in darkness for 10 min at 4 ºC The expression of CD69+CD3+ and CD25+CD3+ on 2342 J. Med. Plant. Res.

Table 1. The expression of surface markers on human T lymphocyte was shown. After treated with 45 µg/ml of PHA in the presence or absence of cucurbitacin B at various concentrations for 24 h, the expressions of surface markers on human T lymphocyte were analysed by flow cytometry.

CD69+CD3+ CD25+CD3+ Type of activations µg/ml Mean ± SD (n=5) (%) Mean ±SD (n=5) (%) Cell control 8.72 ± 2.73 3.62 ± 1.56 PHA 45 77.19 ± 4.14 58.41 ± 6.28 PHA+CUR 0.05 79.75 ± 3.17 51.74 ± 8.43 PHA+CUR 0.25 20.71 ± 11.38* 3.03 ± 1.16* PHA+CUR 0.5 15.66 ± 6.78* 2.83 ± 1.17*

*P < 0.0001, when compared to PHA stimulation group or PHA+0.05 µg/ml of cucurbitacin B. CUR= Cucurbitacin B.

Figure 1. The activated T lymphocyte, CD69+CD3+ (A) and CD25+CD3+ (B) after stimulated with 45 µg/ml of PHA and in the presence of different concentration of cucurbitacin B were shown. Data were expressed by mean ± SD, n=5. The results showed that cucurbitacin B could inhibit the expression of CD69+CD3+ (A) and CD25+CD3+ (B) after 24 h of incubation. (The significantly inhibition when compared between the presence of 0.25 and 0.5 µg/ml cucurbitacin B to PHA stimulation only, *P < 0.0001).

T cells induced by PHA in the presence or absence of after treated with PHA+0.05 µg/ml of cucurbitacin B were curcubitacin B at various concentrations for 24 h is shown 79.75 or 51.74%, respectively. in Table 1 and Figure 1. The results showed that PHA could activate the high expression of CD69+CD3+ (77.19%) and CD25+CD3+ (58.41%). In the group of TNF-ααα assay by ELISA PHA+0.5 µg/ml of cucurbitacin B treated cells, the expression of both CD69+CD3+ (15.66% VS 77.19%, P < TNF-α detection from a cultured medium of PBMC using 0.0001) and CD25+CD3+ (2.83% VS 58.41%, P < ELISA is shown in Figure 2. The release of TNF-α (mean 0.0001) were down-regulated significantly compared to ± SEM) from PHA-stimulated cells after 24, 48, and 72 h those that were stimulated only with PHA after 24 h of of incubation were 1438.61 ± 598.44, 3981.87 ± 323.34 incubation. The expression of CD69+CD3+ (20.71%) and and 3650.51 ± 572.95 pg/ml, respectively (n=3), which CD25+CD3+ (3.03%) after being activated with were significantly higher than the unstimulated control PHA+0.25 µg/ml of cucurbitacin B decreased significantly group (28.54 ± 9.98, 39.92 ± 8.05 and 35.07 ± 4.70, P < compared to PHA (P < 0.0001). No significant difference 0.0001), respectively. The amount of TNF-α, which was was revealed when comparing the surface markers released from the PBMC treated with PHA in the between the groups treated with PHA+0.05 µg/ml of presence of 0.05, 0.25 and 0.5 µg/ml of cucurbitacin B cucurbitacin B to those that were stimulated only with after 48 h, was 980.67 ± 203.39, 770.12 ± 180.83, and PHA. The expression of CD69+CD3+ or CD25+CD3+ 398.53 ± 50.81 pg/ml respectively. These decreased U-pratya et al. 2343

Figure 2. TNF-α detection from cultured medium of human lymphocyte by ELISA was demonstrated by mean ± SEM, (n=3). The MNCs were cultured with 45 µg/ml PHA in the absence or presence of 0.05, 0.25 and 0.5 µg/ml cucurbitacin B compared to unstimulated control for 24 (A), 48(B) and 72 (C) hours respectively. The results showed that cucurbitacin B could inhibit the releasing of TNF-α after 48 and 72 h of PHA incubation. (The significantly inhibition when compared the groups that incubated with cucurbitacin B to PHA stimulation only, *P < 0.0001, **P=0.0001, and ***P=0.0002).

significantly compared to PHA stimulation (3981.87 ± were shown in Table 2 and Figure 4. After 24 h of 323.34, P < 0.0001) after 48 h (Figure 2B). The same incubation with cucurbitacin B, the cytolytic activity was pattern of the release of TNF-α after 72 h was significantly decreased from the control (mean ± SEM, demonstrated (Figure 2C). No difference in TNF-α level 9.83 ± 2.68 vs 40.63 ± 8.30, P = 0.0033) or PHA (9.83 ± after 24 h of incubation was shown (Figure 2A). 2.68 vs 70.09 ± 9.48, P < 0.0001). Forty eight hours of incubation showed the same results as 24 h, as shown in Figure 4. Cucurbitacin B stimulated CTL function was The cytotoxic lymphocyte function assays significantly lower than that stimulated with PHA or even the unstimulated control group. The total CTL activity was performed after collecting PBMC from 10 normal healthy donors. The dot plot analysis from 1 of 10 healthy volunteers with the same DISCUSSION AND CONCLUSION results was demonstrated in Figure 3. After the cells were treated without stimulation (A, B), with PHA (C, D) and The present study investigated the effect of cucurbitacin with cucurbitacin B (E, F) for 48 h, the total CTL function B, a Thai herbal plant, isolated from T. cucumerima L., on was determined as the summation of three cells types, human PBMC, including the activation lymphocyte, the early apoptotic cell (Annexin V+7AAD-), late apoptotic TNF-α secretion and lymphocyte cytotoxicity. cell (Annexin V+7AAD+) and necrotic cell (Annexin V- Cucurbitacin B was found to inhibit the stimulation effect 7AAD+). The total cytolytic activity after PBMC was of PHA on human PBMC not only the expression of treated with cucurbitacin B or PHA for 24, 48 and 72 h surface markers CD69 and CD25 but also the production 2344 J. Med. Plant. Res.

Figure 3. CTL assay of lymphocyte treated with cucurbitacin B compared to control or PHA at 48 h of incubation was shown. Total cytolytic activity of lymphocyte after 48 h of cucurbitacin B incubation was significantly decreased from control or PHA (* compared with control, ** Compared with PHA. n=10).

Table 2. The total cytolytic activity was performed after collection of PBMC from 10 normal healthy donors. Cells were treated with 0.5 µg/ml of cucurbitacin B or 45 µg/ml of PHA for 24, 48 and 72 h

% total cell cytolytic activity at various time Agent 24 h (n=8) 48 h (n=10) 72 h (n=6) Mean ± SEM Mean ± SEM Mean ± SEM Control 40.63 ± 8.30 d 52.49 ± 7.65 a 64.58 ± 11.23 PHA 70.09 ± 9.48 80.73 ± 6.08 79.79 ± 6.00 CUR 9.83 ± 2.68 a 11.89 ± 2.21 b 14.75 ± 2.14 c

aLower significant compared to control (P = 0.0033) or PHA (P < 0.0001), bLower significant compared to control (P<0.0001) or PHA (P<0.0001), cLower significant compared to control (P = 0.0014) or PHA (P < 0.0001), dLower significant compared to PHA (P=0.0347), eLower significant compared to PHA (P<0.0001). CUR = cucurbitacin B

of TNF-α. It is known that the activation of lymphocytes Yokoyama., 1991; Cambiaggi et al., 1992; Borrego et al., expresses some markers such as CD69 and CD25 which 1999). CD25 is named as interleukin-2 receptor (IL-2R) are not expressed or expressed at very low levels at the which is the intermediate activation antigen of T- resting stage (Ziegler et al., 1994; Theze et al. 1996). The lymphocytes. This antigen plays an important role in human CD69 antigen is one of the earliest cell surface lymphocyte proliferation maturation and differentiation markers induced in resting T-lymphocytes and Natural (Theze et al., 1996; Martin-Romero et al., 2000). This Killer (NK) cells following their activation by mitogens, cy- suggests that cucurbitacin B could strongly inhibit the tokines or contact with target cells (Ziegler et al., 1994; immune response of human lymphocytes via the down Testi et al., 1989, 1994; Craston et al., 1997). This regulation of CD69. TNF-αare contributed in the marker is rapidly induced not only in peripheral T cells but regulation of immune cells including the induced also in B cells and NK cells (Risso et al., 1989; Phillips et apoptotic cell death, thymocyte proliferation and al., 1989; Lanier et al., 1998; Gerosa et al., 1991). Once inflamation. This also released from activated expressed on T or NK cells, CD69 acts as a co- CD69+ T-lymphocytes and NK cells (Testi et al., 1989; stimulatory molecule leading to cell proliferation, Santis et al., 1992; Borrego et al., 1999). The reduction of secretion and cytotoxicity, functioning as a signal- TNF-α was found after treating the PHA-stimulated transmitting receptor (Moretta et al., 1991; Karlhofer and PBMC with cucurbitacin B. The reason is due to the down U-pratya et al. 2345

Figure 4. Dot plot analysis from 1 of 10 healthy volunteers with the same results was demonstrated. After cells were treated without stimulation (A, B), with PHA (C, D) or cucurbitacin B (E, F) for 48 hours, the CTL function of lymphocyte was determined, There was significantly lower stimulation between the group of cucurbitacin B compared to normal control (without cucurbitacin B), or PHA group.

regulation of CD69+ cells. Previous studies found that system. The cytolytic activity on K562 cell line of human cucurbitacin B had significant anti-inflammatory activity lymphocytes (cytotoxicity function) was suppressed by (Peter et al., 1997; Agil et al., 1999) and other cucurbitacin B. This may involve the down regulation of cucurbitacins, cucurbitacin R, could inhibit the production CD69 that acts as a co-stimulatory molecule leading to of TNF-α (Escandell et al., 2007). Cucurbitacin B may cell proliferation, secretion, and cytotoxicity. This provides play an importance role in the suppression of immune new evidence for the inhibitory effect of cucurbitacin B on 2346 J. Med. Plant. Res.

human lymphocytes. assay of NK-cell-mediated cytotoxicity in K562 cells revealing three In summary, this study has indicated that cucurbitacin sequential membrane impairment steps using three-color flow- cytometry. J. Immunol. Methods, 307: 41-53. B has immunosuppressive effects on the activation of Kummalue T, Jiratchariyakul W, Srisapoomi T, Sukapanichnant S, Hara human PBMC. The expression of CD69 and CD25 on T, Tani K (2009). Antiproliferative effect of cucurbitacin B extracted activated T-lymphocytes was down regulated and the from Trichosanthes cucumerina L. on human cancer cell lines. Siriraj α Med. J., 61(2): 75-77 production of TNF- was also inhibited. The lymphocyte Lanier L L, Buck DW, Rhodes L, Ding A, Evans E, Barney C, Phillips JH cytotoxicity function was inhibited by cucurbitacin B. (1998). Interleukin 2 activation of natural killer cells rapidly induces Further study of the immunosuppressive effects of the expression and phosphorylation of the Leu-23 activation antigen. cucurbitacin B could well point the way to new and J. Exp. Med., 167: 1572-1585. effective therapy for inflammatory and autoimmune Lyons AB, Parish CR (1994). Determination of lymphocyte division by flow cytometry. J. Immunol. Methods, 171: 131-137. diseases. Marim L, Minguela A, Torio A, Moya-Quiles MR, Muro M, Montes-Ares O, Parrado A, Alvarez-Lopez DM, Garcia-Alonso AM (2003). Flow cytometry quantification of apoptosis and proliferation in Mixed ACKNOWLEDGEMENTS lymphocyte culture. Cytometry Part, 51: 107-118. Martin-Romero C, Santos-Alvarez J, Goberna R, Sanchez-Margalet V (2000). Human leptin enhances activation and proliferation of human The authors special thank Professor Surapol Issaragrisil, circulating T-lymphocytes. Cell Immunol., 199(1): 15-24. Head of Division of Hematology, Department of Medicine Mcmakin PD (1988). A field guide to the flowering plants of Thailand. Siriraj Hospital, for the instrument providing. The authors White Lotus Co. Ltd., Bangkok, Thailand, p. 224. Mitsuyama K, Sata M, Rose-John S (2006). Interleukin-6 trans-signaling would also like to thank Professor Sathit Pichyankul, in inflammator bowel disease. Cytokine Growth Factor Rev., 17(6): Immunology and Vaccinology Research Program, 451-461. Department of Immunology and Medicine, USAMC- Moretta A, Poggi A, Pende D, Tripodi G, Orengo AM, Pella N, AFRIMS, Thailand for his advice and guidance. We thank Augugliaro R, Bottino C, Ciccone E, Moretta L (1991). CD69- mediated pathway of lymphocyte activation: anti-CD69 monoclonal Miss Monraudee chanchai for her kindly prepared plant antibodies trigger the cytolytic activity of different lymphoid effector preparation and also thank Mr. Thomas Radzienda for his cells with exception of cytolytic T lymphocytes expressing T cell kindly proofreading of the manuscript. This study was receptor . J. Exp. Med., 174: 1393-1398. Nefedova Y, Nagaraj S, Rosenbauer A, Muro-Cacho C, Sebti SM, supported by grants from the Thailand Research Fund. Gabrilovich DI (2005). Regulation of dendritic cell differentiation and antitumor immune response in cancer by pharmacologic-selective inhibition of the janus-activated kinase 2/signal transducers and REFERENCES activators of transcription 3 pathway. Cancer Res., 65(20): 9525- 9535. Aggarwal BB, Sethi G, Ahn KS, Sandur SK, Pandey MK, Pattanapanyasat K, Kyle ED, Tongtawe P, Yongvanitchit K, Fucharoen Kunnumakkara AB, Sung B, Ichikawa H (2006). Targeting signal S (1994). Flow cytometric immunophenotyping of lymphocyte subsets transducer and activator of transcription-3 for prevention and therapy in sample that contain a high proportion of non-lymphoid cells. of cancer: modern target but ancient solution. Ann. NY Acad. Sci., Cytometry, 18: 1-10. 1091: 151-169. Peters RR, Saleh TF, Lora M, Patry C, de Brum-Femandes AJ, Far MR, Borrego F, Robertson MJ, Ritz J, Pena J, Solana R (1999). CD69 is a Ribeiro-do-Valle RM (1999). Anti-inflammatory effects of the products stimulatory receptor for natural killer cell and its cytotoxic effect is from Wilbrandia ebracteata on carrageenan-induced pleurisy in mice. blocked by CD94 inhibitor receptor. Immunol., 97: 159-165. Life Sci., 64: 2429-2437. Cambiaggi C, Scupoli MT, Cestari T, Gerosa F, Carra G, Tridente G, Phillips JH, Takeshita T, Sugumura K, Lanier LL (1989). Activation of Accolla RS (1992). Constitutive expression of CD69 in interspecies T- natural killer cells via the p75 interleukin 2 receptor. J. Exp. Med., cell hybrids and locus assignment to human chromosome 12. 170: 291-296. Immunogenetics, 36(2): 117-120. Recio MC, Prieto M, Bonucelli M, Orsi C, Manez S, Giner RM, Cerda- Craston R, Kon M, Mc Dermott A, Ray N, Prentice HG, Lowdell MW Nicolas M, Rios JL (2004). Anti-inflammatory activity of two (1997). Temporal dynamics of CD69 expression on lymphoid cells. J. cucurbitacins isolated from Cayaponia tayuya roots. Planta Med., Immunol. Methods, 209: 37-45. 70(5): 414-420. Derby E, Reddy V, Kopp W, Nelson E, Baseler M, Sayers T, Malyguine Risso A, Cosulich ME, Rubartelli A, Mazza MR, Bargellesi A (1989). A (2001). Three color flow cytometric assay for the study of the MLR3 molecule is an activation antigen shared by human B, T mechanisms of cell mediated cytotoxicity. Immunol. Lett,, 78: 35-39. lymphocytes and T cell precursors. Eur. J. lmmunol., 119: 323-328. Escandell JM, Recio MC, Manez S, Giner RM, Cerda-Nicolas M, Rıos Rungeler P, Castro V, Mora G, Goren N, Vichnewski W, Pahl HL, JL (2007). Cucurbitacin R Reduces the and Bone Merfort I, Schmidt TJ (1999). Inhibition of transcription factor NF- Damage Associated with Adjuvant Arthritis in Lewis Rats by kappa B by sesquiterpene lactones: A proposed molecular Suppression of Tumor Necrosis Factor-∝ in T Lymphocytes and mechanism of action. Bioorg. Med. Chem., 7: 2343-2352. Macrophages. J Pharmaco Exp. Ther., 320(2): 581-590. Sagium P (1979). Thailand Plants. Kasembunnakit Company Limited, Gerosa F, Tommasi M, Scardoni M, Accolla RS, Pozzan T, Libonati M. Bangkok. Tridente G, Carra G (1991). Biochemical and functional analysis of Santis AG, Campanero MR, Alonso JL, Tugores A, Alonso MA, Yagüe the CD69 early activation antigen by two monoclonal antibodies E, Pivel JP, Sánchez-Madrid F (1992). Tumor necrosis factor-a directed to different epitopes. Mol. Immunol., 28: 159-168. production induced in T lymphocytes through the AIM/CD69 Jiratchariyakul W, Mongkarndi P, Theppeang K, Sethajintanin I, activation pathway. Eur. J. Immunol., 22: 1253-1259. Jaridasem S, Frahm AW (1999). Cytotoxic principles from Testi R, D’Ambrosio D, De Maria R, Santoni A (1994). The CD69 Trichosanthes cucumerina L. Thai. J. Phytopharm., 6(2): 1-9. receptor: a multipurpose cell-surface trigger for hematopoietic cells. Karlhofer FM, Yokoyama WM (1991). Stimulation of murine natural killer Immunol. Today, 15: 479-483. (NK) cells by a monoclonal antibody specific for NK1.1 antigen. IL-2- Testi R, Phillips JH, Lanier LL (1989). Leu 23 induction as an early activated NK cells posses additional specific stimulation pathway. J. marker of functional CD3/T cell antigen receptor triggering: Immunol., 146: 3662-3673. Requiredment for receptor cross-linking, prolonged elevation of Kasatori N, Ishikawa F, Ueyama M, Urayama T (2005). A differential intracellular (Ca2+) and stimulation of protein kinase. J. Immunol., 142: U-pratya et al. 2347

1854-1860. Yesilada E, Tanaka S, Sezik E, Tabata M (1988). Isolation of an anti- Tien Dat N, Ji X, Hong YS, Lee JJ (2010). An isoaurone and other inflammatory principle from the fruit juice of Ecballium elaterium . J. constituents from Trichosanthes kirilowii seeds inhibit hypoxia- Nat. Prod., 51: 504-508. inducible factor-1 and nuclear factor-kB. J. Nat. Prod., 73: 1167–1169. Ziegler SF, Ramsdell F, Alderson MR (1994). The activation antigen Theze J, Alzari PM, Bertoglio J (1996). Interleukin-2 and its receptors: CD69. Stem Cells, 12: 456-465. Recent advances and new immunological functions. Immunol. Today. 10: 481-486.