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Original Paper

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

Effect of Sauropus androgynus Leaf Extracts on the Expression of Prolactin and Oxytocin Genes in Lactating BALB/C Mice

SusanSoka HerlinaAlam Stefiani NovaliaBoenjamin TanW.Agustina MaggyT.Suhartono

Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, Jakarta , Indonesia

mice and was predicted to correlate with papaverine con-tent, which is only detected in mature S. androgynus leaves at a concentration of 0.38 8 0.04 g ml 1 .

Copyright 2010 S. Karger AG, Basel

Introduction

Human breast milk is thought to be the best form of nutrition for neonates and infants. The properties of hu-man 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 crit-ical 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 indepen-dently on different cellular receptors, their combined ac-tions 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 androgy-nus , also known in Indonesia as daun katuk ( fig. 1 ).

Key Words Papaverine Prolactin Oxytocin Sauropus androgynus qRT-PCR

Abstract Sauropus androgynus is traditionally consumed by Indone-sians and is believed to increase breast milk production dur-ing lactation. Lactation, a process of milk synthesis and se-cretion, 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 us-ing 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 lac-tating mice was fed a diet without S. androgynus leaf ex-tracts. 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 in-creased the expressions of both genes 15.75- and 25.77-fold, respectively, compared to the control group. The result sug-gested that mature S. androgynus leaf extracts significantly increased the expressions of both genes in lactating BALB/C

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

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

2010 S. Karger AG, Basel16616499/10/00310031$26.00/0

Accessible online at:www.karger.com/jnn

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Soka/Alam/Stefiani/Boenjamin/Agustina/Suhartono

J Nutrigenet Nutrigenomics 2010;3:3136 32

S. androgynus is a shrub grown in some tropical regions, and the leaves of this plant are treated as a common nu-tritious 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] com-pared the lutein and zeaxanthin contents in S. androgy-nus , West Indian pea tree leaves and drumstick tree leaves. They reported that these 3 leafy vegetables con-tained significantly higher amounts of lutein compared to the other vegetables in the region. Ching and Mo-hamed [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 con-tain 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 ap-plied directly to blood vessels.

In this study, papaverine content in the extract of young and mature S. androgynus leaves were quantified.

Additionally, the gene expressions of prolactin and oxy-tocin 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, count-ed 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.

Extraction Solvent A mixture of 85% (v/v) dichloromethane and 15% (v/v) isopro-

panol 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 so-

lution was used to prepare standard working solutions of 1.0, 0.5 and 0.25 g ml 1 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 pe-riod 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 (MF TM , Ireland). Filtrates were transferred to a LiChrolut RP-18, soaked for 15 min and then elut-ed 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.

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

Nutrigenomics of Sauropus androgynus J Nutrigenet Nutrigenomics 2010;3:3136 33

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). Micewere maintained in a single cage with free access to food and wa-ter. 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 ex-tracts, the 2nd group received S. androgynus mature leaf extracts and the 3rd group (the control group) did not receive any S. an-drogynus leaf extracts. S. androgynus leaf extracts were adminis-tered by oral gavage every morning. The given dosage of each leaf extracts was 173.6 mg kg 1 . On the 12th day, all mice were eutha-nized, 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.

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 in-cubated 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 tempera-ture 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

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 add-ing 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 quan-tity of RNA were determined by measuring A260/230 and A260/280 value using NanoDrop 2000 (Thermo Fisher Scientific, USA).

qRT-PCR qRT-PCR was performed in an iQ5 Real-Time Detection Sys-

tem (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 l 1 ), 0.75 l reverse primer (10 pmol l 1 ), 8 l nuclease-free water, 2.5 l RNA template (50 ng l 1 ) 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 data-base (http://www.ncbi.nlm.nih.gov).

Table 1. qPCR conditions

Steps Temperature Time Repeat(s)

cDNA synthesisRT inactivation

50 C95 C

10 min5 min 1

Cycle denaturationPrimer annealing/extension

95 C62 C (oxytocin)/52.5 C (prolactin)/46.7 C (-actin)

10 s30 s 40

Dissociation curve

95 C55 C

1 min1 min 1

Melt curve 55 C (increasing by half degree each cycle) 10 s 81

Target gene Accession No. Primers

Prolactin NM_011164 F: 5-AGG CCT ATC CTG AAG CCA AAG GAAR: 5-TTG TCA ACC TTG TGG GAA TGC CTG

Oxytocin NM_011025 F: 5-TCA CCT ACA GCG GAT CTC AGA CTR: 5-GGG GCA GTT CTG GAT GTA GCA

-Actin NM_007393.3 F: 5-GCT GCG TTT TAC ACC CTT TCTR: 5-TGC TCC AAC CAA CTG CTG TC

Table 2. Primer sequences used forqRT-PCR



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

Results and Discussion

Quantification of Papaverine from S. androgynus Leaves Papaverine hydrochloride solution was used as a stan-

dard 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 ml 1 , 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 so-lution. 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) di-chloromethane and 15% (v/v) isopropanol, and analyzed by HPLC. The concentration of papaverine from S. an-drogynus leaves was only detected in the mature leaves

sample with average concentrations of 0.38 8 0.04 g ml 1 ( table4 ).

Wink [8] reported that papaverine is a secondary me-tabolite 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] re-ported 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 ex-pressions of both prolactin and oxytocin genes in lactat-ing mice supplemented with young S. androgynus leaf ex-

Table 3. HPLC determination of papaverine hydrochloride stan-dard solution

Concentration, g ml1 Time, min Area, mAU Average area

0.00 0.000 0.0 0.00

0.25 4.625 15.3 14.404.640 13.5

0.50 4.689 29.8 30.154.683 30.5

1.00 4.641 68.8 56.354.653 43.9

C orrelation coefficient = 0.998.

Table 4. Concentration of papaverine extracted from S. androgy-nus mature leaves

Time, min Area, mAU Papaverine concentration, g ml1

4.719 17.3 0.3374.693 18.2 0.3554.685 21.8 0.4274.674 20.7 0.4054.681 19.0 0.371

A verage papaverine = 0.38 8 0.04 gml1.

0

5

10

15

20

25

30

Geneexpressionlevel (-fold)

Control

Young leaf extract

Mature leaf extract

Prolactin Oxytocin

Fig. 2. The expression level of prolactin and oxytocin gene in lac-tating BALB/C mice that were given young and mature S. androg-ynus leaf extracts compared to the control group.

Nutrigenomics of Sauropus androgynus J Nutrigenet Nutrigenomics 2010;3:3136 35

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 signif-icantly 15.75- and 25.77-fold when compared to the con-trol 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 ex-pression levels in the group of mice supplemented with mature S. androgynus leaf extracts. The presence of pa-paverine, 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 microsur-gery [10] . On the other hand, papaverine is also a vasodi-lator, 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 regu-lated by dopamine, which acts on D 2 receptors and inhib-its cAMP signaling via G-mediated inactivation of ade-nylyl cyclase [11] . DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of M r 32 kDa, is a major target for the cAMP signaling cascade. Phosphorylation at Thr34 by protein kinase A converts DARPP-32 into a po-tent inhibitor of the wide spectrum protein phospha-tase-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 chan-nels. Mice lacking DARPP-32 are deficient in their mo-lecular, electrophysiological and behavioral responses to dopamine, drugs of abuse and antipsychotic medication, indicating an essential role for DARPP-32 in dopaminer-gic signaling. Dopaminergic signaling is controlled by phosphodiesterases, which degrade cAMP and downreg-ulate cAMP signaling. The inhibition of PDE10A by pa-paverine-activated cAMP/PKA signaling leads to the in-hibition of dopamine D 2 receptor signaling [12] . Thus,

treatment with papaverine can stimulate prolactin re-lease by blocking dopamine receptors.

The other component of S. androgynus that could af-fect increasing of milk production is sterol [13] . The nu-trients in S. androgynus could also enhance the milk pro-duction by increasing the activity of glucose metabolism for the synthesis of lactose [14] . S. androgynus leavescontain 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 ml 1 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 ex-tracts increased 9.04- and 2.25-fold, respectively, com-pared to the control group. On the other hand, the ex-pressions of prolactin and oxytocin genes in mice supple-mented with mature S. androgynus leaf extracts increased 15.75- and 25.77-fold, respectively, compared to the con-trol 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.

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