Original Paper

J Nutrigenet Nutrigenomics 2010;3:3136 DOI: 10.1159/000319710

Received: June 8, 2010 Accepted: July 27, 2010 Published online: August 26, 2010

Effect of Sauropus androgynus Leaf Extracts on Free Author Copy for perthe Expression of Prolactin and Oxytocin Genes in sonal use only Lactating BALB/C Mice ANY DISTRIBUTION OF THIS
SusanSoka HerlinaAlam Stefiani MaggyT.Suhartono
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Key Words Papaverine Prolactin Oxytocin Sauropus androgynus qRT-PCR

mice and was predicted to correlate with papaverine content, which is only detected in mature S. androgynus leaves at a concentration of 0.38 8 0.04 g ml1.

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Abstract Sauropus androgynus is traditionally consumed by Indonesians and is believed to increase breast milk production during lactation. Lactation, a process of milk synthesis and secretion, occurs with the help of 2 hormones, prolactin and oxytocin. The expressions of genes encoding prolactin and oxytocin were analyzed in lactating BALB/C mice brains using qRT-PCR. A total of 24 lactating BALB/C mice were fed with experimental diets for 12 days. Two groups of lactating mice were fed with diets containing either young or mature S. androgynus leaf extracts. For the control, one group of lactating mice was fed a diet without S. androgynus leaf extracts. Supplementation of young S. androgynus leaf extracts increased the expression of prolactin and oxytocin genes in lactating mice 9.04- and 2.25-fold, respectively. Meanwhile, supplementation of mature S. androgynus leaf extracts increased the expressions of both genes 15.75- and 25.77-fold, respectively, compared to the control group. The result suggested that mature S. androgynus leaf extracts significantly increased the expressions of both genes in lactating BALB/C

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Introduction

Human breast milk is thought to be the best form of nutrition for neonates and infants. The properties of human milk facilitate the transition of life from in utero to ex utero. These dynamic fluids provide a diverse array of bioactive substances to the developing infant during critical periods of brain, immune and gut development. The cyclical process of milk synthesis and secretion, termed as lactation, occurs with the help of 2 hormones, prolactin and oxytocin. While prolactin and oxytocin act independently on different cellular receptors, their combined actions are essential for successful lactation [1]. A survey in Indonesia reported that 38% of mothers stopped breastfeeding because of a lack of breast milk production [2]. As a result, many traditional supplements believed to increase humans breast milk production are offered on the market. One of them is Sauropus androgynus, also known in Indonesia as daun katuk (fig. 1).
Susan Soka, MSc Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia Jl. Jenderal Sudirman No.51 Jakarta 12930 (Indonesia) Tel. +62 21 573 1740, Fax +62 21 571 9060, E-Mail susan.soka@atmajaya.ac.id

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Additionally, the gene expressions of prolactin and oxytocin hormones in lactating BALB/C mice supplemented with both extracts were compared.
Material and Methods
Raw Material S. androgynus leaves were purchased from a local market in Jakarta, brought to the laboratory, and freeze-dried after some preparations. S. androgynus leaves were categorized as young leaves or mature leaves. The young leaves were the first 3 main leaves counted from the peak and were 1.0 cm in width and 2.5 cm in length. The young leaves were bright green in color. The mature leaves were picked at the 8th, 9th and 10th leaves, counted from the peak. These mature leaves were 2.0 cm in width and 5.0 cm in length, and they were dark green. Freeze Drying The leaves were washed with water and put on small trays. The trays were covered with aluminum foil and placed into the freeze drier (Alpha 24 LD plus; Christ) for 48 h. The dried leaves were pulverized using a blender and kept at 4 C.

Fig. 1. Sauropus androgynus, also known as daun katuk in Indo-

nesia.

S. androgynus is a shrub grown in some tropical regions, and the leaves of this plant are treated as a common nutritious vegetable in Asia. These leaves are traditionally used by mothers in Indonesia to increase their breast milk production. Many research works have been conducted to assess the vitamin contents of S. androgynus. Liu et al. [3] compared the lutein and zeaxanthin contents in S. androgynus, West Indian pea tree leaves and drumstick tree leaves. They reported that these 3 leafy vegetables contained significantly higher amounts of lutein compared to the other vegetables in the region. Ching and Mohamed [4] compared -tocopherol content in 62 edible tropical plants, and the result showed that the highest tocopherol content was in S. androgynus. According to Saroni et al. [2], S. androgynus leaf extracts increased breast milk production up to 50.7%. S. androgynus leaves were previously reported to contain a considerable amount of the alkaloid papaverine up to 580 mg per 100 g of fresh leaf [5]. Papaverine has been approved to treat spasms of the gastrointestinal tract, bile ducts and ureter. It is also used as cerebral and coronary vasodilators. Additionally, it has been used as a smooth muscle relaxant in microsurgery, where it is applied directly to blood vessels. In this study, papaverine content in the extract of young and mature S. androgynus leaves were quantified.
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Extraction Solvent A mixture of 85% (v/v) dichloromethane and 15% (v/v) isopropanol was used as extraction solvents. This solvent was also used to elute the sample. Papaverine Hydrocloride Standard Solution A papaverine hydrochloride (1 mg/10 ml methanol) stock solution was used to prepare standard working solutions of 1.0, 0.5 and 0.25 g ml1 working solutions. These solutions were mixed with citric buffer at pH 6.5 at ratio 1: 1 (v/v) and filtered through the membrane filters (Pall Corp., USA) with a pore size of 0.20 m. A 20- l portion of the solution was injected on the column by an autosampler and a chromatogram was developed for a period of 10 min [6].

Papaverine Extraction from S. androgynus A total of 200 g powdered S. androgynus was suspended in 25 ml of 2.5% (v/v) acetic acid and was extracted for 20 min by suprasonication. The mixture was centrifuged at 8,500 rpm for 2min and the supernatant was filtered. This step was repeated twice. The filtrates were combined and 2.5% (v/v) acetic acid was added to give a final volume 50 ml. The pH of the solution was then adjusted to pH 9.0 with ammonia (2.5% v/v) and was filtered through a 0.45- m Millipore filter (MFTM, Ireland). Filtrates were transferred to a LiChrolut RP-18, soaked for 15 min and then eluted with dichloromethane-isopropanol (85: 15). The eluate was evaporated to dryness and the residue was dissolved in 2.5 ml methanol [7]. Chromatography The papaverine content was determined on the Agilent 1100 HPLC system using a Zorbax Eclipse XDB-C18 reverse-phase column 30 ! 4.6 mm. The mobile phase, methanol/water (60:40 v/v), was used at 1 ml/min, and the UV detector was set at 278 nm.

Soka/Alam/Stefiani/Boenjamin/ Agustina/Suhartono

Table 1. qPCR conditions

Steps cDNA synthesis RT inactivation Cycle denaturation Primer annealing/extension Dissociation curve Melt curve

Temperature 50 C 95 C 95 C 62 C (oxytocin)/52.5 C (prolactin)/46.7 C ( -actin) 95 C 55 C 55 C (increasing by half degree each cycle)

Time 10 min 5 min 10 s 30 s 1 min 1 min 10 s

Repeat(s) 1 40 1 81

Table 2. Primer sequences used for

qRT-PCR

Target gene Prolactin Oxytocin -Actin

Accession No. NM_011164 NM_011025 NM_007393.3

Primers F: 5 -AGG CCT ATC CTG AAG CCA AAG GAA R: 5 -TTG TCA ACC TTG TGG GAA TGC CTG F: 5 -TCA CCT ACA GCG GAT CTC AGA CT R: 5 -GGG GCA GTT CTG GAT GTA GCA F: 5 -GCT GCG TTT TAC ACC CTT TCT R: 5 -TGC TCC AAC CAA CTG CTG TC

Animal Preparation Mice (Mus musculus), from the strain BALB/C and which were pregnant for the second time were obtained from the Rodentia Facility of PT. Bimana Indomedical (Bogor, Indonesia). Mice were maintained in a single cage with free access to food and water. The treatments were given for 12 days during the lactation period. A total of 24 lactating BALB/C mice were divided into 3 groups of 8. The 1st group received S. androgynus young leaf extracts, the 2nd group received S. androgynus mature leaf extracts and the 3rd group (the control group) did not receive any S. androgynus leaf extracts. S. androgynus leaf extracts were administered by oral gavage every morning. The given dosage of each leaf extracts was 173.6 mg kg1. On the 12th day, all mice were euthanized, and their pituitary glands were collected and stored at 70 C. All procedures were approved by the Animal Care and Use Committee of PT. Bimana Indomedical.

was mixed with 1 volume of 70% (v/v) ethanol, vortexed for 15 s, and transferred to the RNeasy column in a 2-ml tube. The mixture was then centrifuged at 8,000 rpm for 15 s, and the flow-through was discarded. The RNA in the membrane of the RNeasy column was washed with 700 l buffer RW1, centrifuged at 8,000 rpm for 15 s and the flow-through was discarded. Then, it was washed again with 500 l buffer RPE, centrifuged at 8,000 rpm for 15 s and the flow-through was discarded. This step was done twice, with 8,000 rpm centrifugation for 2 s during the 2nd round. Afterwards, the RNeasy column was placed in a new microfuge tube, eluted by adding 30 l RNase-free water and centrifuged at 8,000 rpm for 1 min. The resulting RNAs were stored at 20 C. The quality and quantity of RNA were determined by measuring A260/230 and A260/280 value using NanoDrop 2000 (Thermo Fisher Scientific, USA).

Isolation of mRNA Total mice mRNAs were extracted from pituitary glands using QIAzol reagent (Qiagen, USA) for cell lysis, RNeasy Lipid Tissue Mini Kit (Qiagen) for extraction from lipid tissue and QIAshredder (Qiagen) for purification. Mouse brain samples (^100 mg) were disrupted and homogenized in 2 ml QIAzol lysis reagent, then incubated at room temperature for 5 min. The solution was moved into a new Eppendorf tube. Then 1/5 volume of chloroform was added, shaken vigorously for 15 s and incubated at room temperature for 15 min. The mixture was centrifuged at 12,000 rpm at 4 C for 15 min. The upper aqueous phase was transferred to the QIAshredder and centrifuged at 8,000 rpm for 15 s. The solution

qRT-PCR qRT-PCR was performed in an iQ5 Real-Time Detection System (BioRad, USA) using iScript One-Step RT-PCR Kit with SYBR Green (BioRad). The master mix for qRT-PCR consisted of 12.5 l 2! SYBR Green RT-PCR reaction mix, 0.75 l forward primer (10 pmol l1), 0.75 l reverse primer (10 pmol l1), 8 l nuclease-free water, 2.5 l RNA template (50 ng l1) and 0.5 l iScript reverse transcriptase for 1-step RT-PCR. The conditions for qRT-PCR are described in table1. Primer sequences, specific to oxytocin, prolactin and -actin encoding genes, were designed using the FastPCR program (table2). The housekeeping gene, actin, was used as the reference gene. Full sequences of oxytocin, prolactin and -actin genes were taken from the GenBank database (http://www.ncbi.nlm.nih.gov).

Nutrigenomics of Sauropus androgynus

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30 25 20 15

Table 3. HPLC determination of papaverine hydrochloride standard solution

Control Young leaf extract Mature leaf extract

Concentration, g ml1 0.00 0.25 0.50

Time, min Area, mAU Average area 0.000 4.625 4.640 4.689 4.683 4.641 4.653 0.0 15.3 13.5 29.8 30.5 68.8 43.9 0.00 14.40 30.15 56.35

Gene expression level (-fold)

10

1.00
5 0 Prolactin Oxytocin

Correlation coefficient = 0.998.

Fig. 2. The expression level of prolactin and oxytocin gene in lac-

tating BALB/C mice that were given young and mature S. androgynus leaf extracts compared to the control group.

Table 4. Concentration of papaverine extracted from S. androgynus mature leaves

Time, min Data Analysis Gene expression levels were calculated based on the cycle threshold (Ct) value using the following formulas: Ct (treatment) = Ct (treatment) Ct ( -actin) Ct (control) = Ct (control) Ct ( -actin) Ct = Ct (treatment) Ct (control) Respective gene expression level = 2 Ct 4.719 4.693 4.685 4.674 4.681

Area, mAU 17.3 18.2 21.8 20.7 19.0

Papaverine concentration, g ml1 0.337 0.355 0.427 0.405 0.371

Average papaverine = 0.38 8 0.04 g ml1.

Results and Discussion

Quantification of Papaverine from S. androgynus Leaves Papaverine hydrochloride solution was used as a standard for identification and quantification of the HPLC peak. The linear relationship between the area of the peaks and concentration of papaverine hydrochloride standard solution within the range 0.251.0 g ml1, was obtained using methanol and water (60:40 v/v) as the mobile phase for HPLC method. Table3 represents the retention time and average covered area from each concentration of papaverine hydrochloride standard solution. The correlation coefficient of the calibration curve was 0.998, which confirmed the accuracy of this method. S. androgynus leaves were extracted with 85% (v/v) dichloromethane and 15% (v/v) isopropanol, and analyzed by HPLC. The concentration of papaverine from S. androgynus leaves was only detected in the mature leaves
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J Nutrigenet Nutrigenomics 2010;3:3136

sample with average concentrations of 0.38 8 0.04 g ml1 (table4). Wink [8] reported that papaverine is a secondary metabolite and therefore not detected in the S. androgynus young leaves. These secondary metabolites are synthesized along with the growth of plant. Yoshimatsu et al. [9] reported that various amounts of detectable papaverine from Papaver is related to various extraction solvents, cultivar and the part of the plant used as the source in the research.

Expression of Oxytocin and Prolactin Genes in Lactating BALB/C Mice The expressions of prolactin and oxytocin genes were compared between mice groups supplemented with young and mature S. androgynus leaf extracts, and water as a control during lactating period. The results, as shown in figure 2, indicate that the expressions of both prolactin and oxytocin genes in lactating mice supplemented with young S. androgynus leaf exSoka/Alam/Stefiani/Boenjamin/ Agustina/Suhartono

tracts increased 9.04- and 2.25-fold, respectively, when compared to the control group. On the other hand, the expressions of both genes in lactating mice supplemented with mature S. androgynus leaf extracts increased significantly 15.75- and 25.77-fold when compared to the control group (p ! 0.05). Based on these results, the mouse group that was supplemented with mature S. androgynus leaf extracts had the most significant increment in the expression of prolactin and oxytocin genes compared to other groups. Mature S. androgynus leaves might contain higher secondary metabolites than the younger ones. This could be the reason for the higher prolactin and oxytocin expression levels in the group of mice supplemented with mature S. androgynus leaf extracts. The presence of papaverine, which is one of the secondary metabolites in S. androgynus leaves, might be related to the increased prolactin and oxytocin production. Papaverine inhibits phosphodiesterase activity and accumulates cAMP, which functions as a second messenger for intracellular signal transduction. A high level of cAMP will cause smooth muscles, which surround blood vessels, to relax. Therefore, papaverine is usually used as a muscle relaxant and can be applied directly to blood vessels in microsurgery [10]. On the other hand, papaverine is also a vasodilator, which is an agent that widens the blood vessels. When these vessels dilate, the flow of blood is increased. Therefore, it can help the circulation of prolactin and oxytocin through the bloodstream. The expression of the prolactin-encoded gene is regulated by dopamine, which acts on D2 receptors and inhibits cAMP signaling via G-mediated inactivation of adenylyl cyclase [11]. DARPP-32, a dopamine- and cAMPregulated phosphoprotein of Mr 32 kDa, is a major target for the cAMP signaling cascade. Phosphorylation at Thr34 by protein kinase A converts DARPP-32 into a potent inhibitor of the wide spectrum protein phosphatase-1. The inhibition of protein phosphatase-1 thereby controls the phosphorylation state and activity of many downstream physiological effectors, including various neurotransmitter receptors and voltage-gated ion channels. Mice lacking DARPP-32 are deficient in their molecular, electrophysiological and behavioral responses to dopamine, drugs of abuse and antipsychotic medication, indicating an essential role for DARPP-32 in dopaminergic signaling. Dopaminergic signaling is controlled by phosphodiesterases, which degrade cAMP and downregulate cAMP signaling. The inhibition of PDE10A by papaverine-activated cAMP/PKA signaling leads to the inhibition of dopamine D2 receptor signaling [12]. Thus,
Nutrigenomics of Sauropus androgynus

treatment with papaverine can stimulate prolactin release by blocking dopamine receptors. The other component of S. androgynus that could affect increasing of milk production is sterol [13]. The nutrients in S. androgynus could also enhance the milk production by increasing the activity of glucose metabolism for the synthesis of lactose [14]. S. androgynus leaves contain 88.32 8 0.06 g moisture, 4.84 8 0.19 g protein, 0.19 8 0.03 g lipids, 5.36 8 0.65 g carbohydrates, 1.11 8 0.14 g fiber and 0.17 8 0.08 g ash, 204 mg calcium, 83 mg phosphorus, 2.7 mg iron, 10,370 IU vitamin A, 0.1 mg vitamin B1 and 580 mg papaverine [5, 15, 16]. These nutrients are considered essential for human health and, hence, are good for routine consumption.

Conclusion

The result shows that the concentration of papaverine from S. androgynus leaves was only detected in mature leaves with an average concentration of 0.38 8 0.04 g ml1 and was not detected in the S. androgynus young leaves. The expressions of prolactin and oxytocin genes in mice supplemented with young S. androgynus leaf extracts increased 9.04- and 2.25-fold, respectively, compared to the control group. On the other hand, the expressions of prolactin and oxytocin genes in mice supplemented with mature S. androgynus leaf extracts increased 15.75- and 25.77-fold, respectively, compared to the control group. This research confirmed the positive effect of papaverine as an inducer of the gene expression required for good lactation and therefore supports the current practice of traditional belief in lactation.

Acknowledgements
This work was supported by a grant from the Indonesia Toray Science Foundation 2009 and The Research Institute of Atma Jaya Catholic University of Indonesia.

References

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3 Liu Y, Perera CO, Suresh V: Comparison of three chosen vegetables with others from South East Asia for their lutein and zeaxanthin content. Food Chem 2007; 101: 1533 1539. 4 Ching LS, Mohamed S: Alpha-tocopherol content in 62 edible tropical plants. J Agric Food Chem 2001;49:31013105. 5 Padmavathi P, Prabhakara RM: Nutritive value of Sauropus androgynus leaves. Plant Foods Human Nutr 1990;40:107113. 6 Kasperek R: Determination of diclofenac sodium and papaverine hydrochloride in tablets by HPLC method. Acta Pol Pharm 2008; 65:403408. 7 Krenn L, Glantschnig S, Sorgner U: Determination of the five major opium alkaloids by reverse-phase high-performance liquid chromatography on a base-deactivated stationary phase. Chromatographia 1997; 47: 2124.

8 Wink M: Introduction: biochemistry, role and biotechnology of secondary metabolites. Annu Plant Rev 1999;2:116. 9 Yoshimatsu K, Kiuchi F, Shimomura K, Makino Y: A rapid and reliable solid-phase extraction method for high-performance liquid chromatographic analysis of opium alkaloids from papaver plants. Chem Pharm Bull (Tokyo) 2005;53:14461450. 10 Triner L, Vulliemoz Y, Schwartz I, Nahas GG: Cyclic phosphodiesterase activity and the action of papaverine. Biochem Biophys Res Com 1990;40:6469. 11 Stoof JC, Kebabian JW: Opposing roles for D-1 and D-2 dopamine receptor in efflux of cyclic AMP from rat neostriatum. Nature 1981;294:366368 12 Nishi A, Kuroiwa M, Miller DB, OCallaghan JP, Bateup HS, Shuto T, Sotogaku N, Fukuda T, Heintz N, Greengard P, Snyder GL: Distinct roles of PDE4 and PDE10A in the regulation of cAMP/PKA signaling in the striatum. J Neurosci 2008;28:1046010471.

13 Prajonggo TS, Djatmiko W, Soemarno T, Lunnardi JL: Effect of Sauropus androgynus Merr. on the histology of mammary gland of lactating mice. National Symposium Proceeding ISFI 11, 1990, pp 735739. 14 Suprayogi A, Kusumorini N, Achmadi P: Effect of Sauropus androgynus (L.) Merr on the metabolism, production, and the milk composition of lactating goat. Proceeding Symposium of Research in Natural Medicine 8, 1996, pp 336340. 15 Rukmana HR, Harahap IM: Katuk: Its Potentials and Effects. Yogyakarta, Kanisius, 2003. 16 Benjapak N, Swatsitang P, Tanpanich S: Determination of antioxidant capacity and nutritive values of Pak-Wanban (Sauropus androgynus L. Merr.). KKU Sci J 2008; 36: 279289.

Free Author Copy for personal use only

ANY DISTRIBUTION OF THIS ARTICLE WITHOUT WRITTEN CONSENT FROM S. KARGER AG, BASEL IS A VIOLATION OF THE COPYRIGHT. Written permission to distribute the PDF will be granted against payment of a permission fee, which is based on the number of accesses required. Please contact permission@karger.ch

Free Author Copy for personal use only

ANY DISTRIBUTION OF THIS ARTICLE WITHOUT WRITTEN CONSENT FROM S. KARGER AG, BASEL IS A VIOLATION OF THE COPYRIGHT. Written permission to distribute the PDF will be granted against payment of a permission fee, which is based on the number of accesses required. Please contact permission@karge

36

J Nutrigenet Nutrigenomics 2010;3:3136

Soka/Alam/Stefiani/Boenjamin/ Agustina/Suhartono