Characterization of jec absorbed compounds in lactating rat's milk and suckling neonates

CHARACTERIZATION OF JEC ABSORBED COMPOUNDS IN LACTATING
RAT’S MILK AND SUCKLING NEONATES.
Norhafilda Ismail
Department of Biochemistry, School of Bioscience and Biotechnology,
Faculty of Science & Technology, National University of Malaysia, 43600 Bangi, Malaysia.
Corresponding author: hafildaismail@gmail.com
ABSTRACT
Jamu Ratus, a widespread use of traditional complementary herbal remedies upon post-
partum recovery. The herbal remedies constituents may comprising abundance of herbs and
spices namely Piper sp., Alpinia sp., Kaempferia galangal, Curcuma sp. which representing
the complexity of herbal formulas and the presence of ubiquitous of bioactive compounds.
Preliminary studies has been conducted using different concentrations of therapeutic dosages
as given; 0mg/kg/day as control; 150mg/kg/day; 500mg/kg/day and 1500mg/kg/day in
lactating rat’s milk and suckling neonates after giving birth within 1 month upon gestation
period. The main objectives is to characterize the absorbed of JEC compounds derived from
ethanolic fractions compounds using lactating’ rats milks and plasma tissues from liver and
brain’s (n=30) of suckling neonates. The subjected samples tested were collected, weighed
and further analysed using Thin Layer Chromatography (TLC); High Performance of
Chromatography (HPLC) and Liquid Chromatography Mass Spectrometry (LCMS) using
different phase of solvent as eluents. The presence of flavonoids compounds even at lower
dosage of therapeutic dosage (150mg/kg/day) given in neonatal tissues brain persistent with
time and dosage given; giving good insight in understanding of xenobiotic metabolism in the
liver. The bioaccumulation of absorbed JEC compounds in neonatal brain and liver tissues
showing the bioavailability of the drug itself to retain, persistent with time of drug exposure.
Albeit the vulnerability of the neonatal liver and brain in early stage development upon drug
interference to transverse into the brain tissues; thus, implicated the toxicity potential whilst
exhibits and affecting cognitive development and physiological activities and henceforth
need to be further resolved.
Key Words;, JEC compounds, xenobiotic , TLC, HPLC and LCMS.

Introduction
Jamu Ratus, a widespread use of traditional complementary herbal remedies by Malay
women upon post-partum recovery. Malaysia has a well-developed traditional system of
medicine, which has been in practice use by complementary herbal practitioners for
treatment various ailments due to its pharmacological activities [30].Preliminary scientific
studies has shown extensive research on traditional complementary herbal remedies using
plants versus biosynthesis modern drug towards the efficacy uses of the drug’s itself. The
constituents of herbal remedies may comprised with ubiquitous herbs and spices inherited
from the late ancestor to cure and care after giving birth. The consumption of Jamu Ratus in
daily dietary intake and traditionally being claimed to be able in enhancing the body
temperature of the mother during postnatal and gestation period for the suckling new born
baby. Kaempferol is a polyphenol antioxidant found in fruits and vegetables.
Epidemiological studies have shown an inverse relationship between kaempferol intake and
cancer. Kaempferol may help by augmenting the body’s antioxidant defense against free
radicals, which promote the development of cancer. [1], [13]. Flavonoids may range from
flavones, flavonols, flavonol aglyconMany flavonoids possess in vitro cancer, antiviral and
anti-inflammatory properties that having ability to inhibit broad range of enzymes and to act
as potent antioxidants [9]. Kaempferol is a yellow crystalline solid with a melting point of
276-278°C. It is slightly soluble in water but soluble in hot ethanol and ether. The health
status of using these traditionally prescribed Jamu Ratus seems dubious and need to be
resolved in future investigations. Thus, these led extensively promising studies which were
conducted in order to generate specific useful information that will provide good insight in
the future therapeutic traditional medicines that is safe to be consumed in human (controlled
drug) without giving bad adverse effect towards both party (mother and the suckling infant).

Experimental Designs
Materials and Methods
Jamu Ratus are purchased from local supplier, Johor Bharu, Malaysia in powder form. The
crude JEC, decoctions extracts were made in three different crucial extraction methods.
Firstly, the herbs are weighed , dissolved and defatted with 5 volumes of Petroleum Ether
solvents overnight (40°C-60°C).. Then, the residue were filtered and dried in room
temperature with Whatman 4 paper. The residues were extracted with 10 volume of ethanol
solvents (95%) in 80°C with automatic Soxhlet extraction methods within 6 hours to remove
oil , fat (defatting) and debris from the extracts. Ethanolic extract were dried in vacuo using
rotary evaporator to produce high yield of polar JEC compounds extractions. The residues
ethanolic extracts were undergone partition of chloroform and water with ratio of 1:1
(1.2ml/g of Jamu Ratus). The chloroform partition were done in three time, collected and
dried in vacuo. The percentage of JEC turn over (50µl-100µl) were calculated after
lyophilized with nitrogen gas. The JEC yields distributed in serial glass vials with (mg) for
each, lyophilized with nitrogen gas in 60°C upon JEC treatment. The lyophilized crude JEC
were stored in -30°C to avoid oxidation process [4]. These experimental studies has been
conducted using different batches of relative therapeutic dosages JEC, as such; 0x
(0mg/kg/day) control; 3x (150mg/kg/day); 10x (500mg/kg/day) and 30x (1500mg/kg/day) to
observe the efficacy of the JEC compounds in rat’s model. JEC known as ethanolic extract
and chloroform fraction of Jamu Ratus.
These research has been done for 12 replications of animal models using lactating rats and
suckling neonates (n=144). Two set of animal model, which is 12 lactating rats per set were
divided into four group with three rats per group. The group were divided based on relative
therapeutic dosages 0x (0mg/kg/day) control, 3x (150mg/kg/day), 10x (500mg/kg/day) and
30x (1500mg/kg/day). The lactating rats and suckling neonates were orally fed and
administered daily with crude JEC extracts via force feeding method within 1 months
(chronic studies) during gestation period. The control animal were fed with carboxyl methyl
cellulose solution (CMC) dissolved in saline water. These subjected sample of plasma tissues

were obtained upon post mortem process from (plasma) blood, liver (heparin) and plasma
(blood brain barriers) of the suckling neonates.
Milk sampling,
The milk sampling were done in 5th
and 9th
day of JEC treatment. The milk sampling were
done after one hour of post-drug. The lactating rats were anesthetized with diethyl ether,
injected with oxytocin hormones (2 I.U) intravenously through vein’ tail for each rats to
promote the production of milk. Enhancement of milk production was done by massaging the
mammary glands and collected by using micropipette, and eppendorf tube (1.0ml) and stored
in -30°C for next extraction. The average of milk collections were documented. Blood
plasma were collected on 9th
day after JEC treatment and at the end of milk sampling.
Suckling also known as major stimulus for oxytocin secretion during lactation in the rat [14],
[15]. Frequency of milks ejections rather than the amount of oxytocin per milk ejection has
been found previously to depend, in some circumstances on litter size [13];[23]..
Quantitative and Qualitative Analysis
The identification of the JEC compounds binding to plasma protein of tested samples were
analysed and detected using Thin Layer Chromatography (TLC) techniques , HPLC (High
Performance Liquid Chromatography) and LCMS (Liquid Chromatography Mass
Spectrometry) for reproducible and accurate outcomes.
Thin Layer Chromatography method
The square shaped of glass plate in 20cm X 20cm (length and width) were cleaned with
acetone. 30g of silica gel GF powder are weighed and mixed up with 75ml of distilled water
and homogenized with vortex and spread on the glass plates up to 0.4mm of thickness in a
row. Then, the square plates were dried and preheated in oven up to 110°C for about 30
minutes to activate the silica gel upon being used. The prominent solvent system used are
Chloroform: Acetic Acid (90:10/100ml); (9:1, v/v) of total volume.

Organic and aqueous phase of tested samples were separated and dissolved in chloroform
and methanol with 100µl volume for Thin Layer Chromatography analysis. Then, 25µl of
each extract were spotted on the silica plate. Almost 2.5mg of JEC were spotted on the same
plate as reference. The elution was made until ¾ of TLC plate within 1 hours. The separated
bands were detected under ultraviolet exposure (366nm). The unique band represented in
fluorescence band are expected to be presence in subjected plasma sample pre-treated with
JEC relative therapeutic dosages and absence in control sample (0mg/kg/day) of JEC
treatment.
High Performance Liquid Chromatography (HPLC)
The unique bands were detected on TLC chromatograms of subjected milk samples were
scrapped off, collected and further extracted. The extraction method was done by adding
methanol and chloroform solvent (1:3), then vortex for 1 minute and soaked in ultrasonic
bath (60°C) for about 20 minutes. The mixture were centrifuged in 13000 rpm for about 10
minute.as and supernatant were collected in different vials. These crucial steps were repeated
for three times. The collected supernatant were lyophilized using nitrogen gas in 60°C and
further analysed by adding 1ml of Methanol (HPLC grade) , vortex, and filtered with picagari
filtration (the filtration membrane with diameter 13mm, pores: 0.45 µm ) before analysed
using HPLC and LCMS techniques. 20µL of whole samples for unique fraction and unique
fraction for scrapped TLC chromatogram fraction were injected for HPLC and LCMS
analysis. These methods applied in control and standard samples (kaempferol, quercetin and
JEC).
High Performance Liquid Chromatography (HPLC) analysis were employed to detect the
flavonoid, isoflavonoid and phenolic compounds [5]. The apparatus used was Intelligent
HPLC Pump Jusco PU-980 , connected with Degassex, degasser vacuum DG440 model and
C18 column type Symmetry® 5µm (3.9 x 150mm column).The absorbent detector used was
Waters 484. Two mobile phase are used which are A, formic acid –water 1% (5:95 v/v) and
B ,methanol HPLC grade. The elusion profile are 0-2min , 7% B in A (isocratic): 2-8 min , 7-
15 % B in A (linear gradient): 8-25 min, 15-75 % B in A (linear gradient): 25-27min , 75-

80% B in a (linear gradient); 27-29 min, 80%B in A (isocratic), 7% B in A (isocratic): 29-33
min , 7% B in a (isocratic), 33-35 min with the flow rate 1ml/min. The thermostat
temperature is 20 °C while the column pressure is 81 bar and UV detection system, viewed
under 280nm wavelength. The analysis has been done in laboratory 1125, Department of
Food Technology and Chemistry Science, Faculty of Science and Technology, National
University of Malaysia, UKM, Bangi, Selangor.
Liquid Chromatography Mass Spectrometry (LCMS)
Liquid Chromatography Mass Spectrometry (LCMS) modified analyses applied to be used
in order to optimize the detection and characterization of JEC components the JEC absorbed
components of subjected samples upon lactating dams tissues and suckling neonates.
Through the high throughput analyses, the molecular weight of absorbed JEC compounds
were able to be identified based on comparison with elution time RT value, mass spectra of
spectrometry compounds of control samples, kaempferol and quercetin standard samples
with samples pretreated with JEC relative therapeutic JEC dosages.
LCMS (Liquid Chromatography Mass Spectrometry analyses were employed using
microTOF-Q 86 connected with Agilent 1100 HPLC, Gilson321 Pump with Injector Auto
sampler and Jupiter 5u C 18 300A column 5 µm (2.0 x 250mm column). Absorbent Derector
used was Waters 484. Two mobile phase used in these analyses; A: Formic acid-water 1%
(5:95) and B, methanol HPLC grade. Elution profile is 0-2min, 7% B in A (isocratic); 2-8
min, 7-5 % B in A (linear gradient); 8-25 min, 15-75% B in A (linear gradient); 25-27 min,
75-80% B in A (linear gradient); 27-29 min, 80% B in A (isocratic) with flow rate fluorate
0.2ml/min. Thermostat temperature is 20°C while column pressure 81 bar and the UV ,
ultraviolet detection in 280nm. Nitrogen gas are used to break down the ionic fragment (in 80
°C, 40V) to produce ion products (in 160°C, 2V). These analysis was done in ToF laboratory
of Chemistry Building, Centre of Research and Innovation Management, Faculty of Science
and Technology, National University of Malaysia, UKM, Bangi, Selangor, Malaysia.

Results and Discussions
The flavonoid-binding protein plasma from subjected plasma blood, liver and brain of
suckling neonates were then being detected using TLC (Thin Layer Chromatography)
classical analysis for bioactive compounds in plant [11],[18] which inferred the absorbed
JEC compounds in tissues. The separation techniques shown the presence of yellowish and
green fluorescence band persistent with time and therapeutic dosages given. This may
implying the presence of secondary metabolite of the flavonoid compounds (flavonol,
flavonol aglycone, alkaloid, phenolic acids) derived from JEC absorbed tissues samples
tested compared to control samples.
The sampling data were analysed and showing the prominent presence of flavonoid binding
protein based on fluorescence bands colours appeared (yellowish and green bands) eluted
with Rt (0.86) prominent and persistent in highest therapeutic dosage (1500mg/kg/day) prior
to 1 month of JEC treatment under ultraviolet (UV) light exposure (366nm) in whole milk
and liver of lactating dam’s samples. The yellowish and green band are known as polar
compound , depicted out and emanated from tested plasma sample seems to be present and
eluted farther than origin point using Thin Layer Chromatography (TLC) classical screening
and separation analysis, [11], [18]. The fluorescence band were scrapped out, weighed and
further analyses using HPLC analysis. The polar compounds are eluted farther than origin
point using main eluents mobile phase, good resolution of separation (Chloroform: Acetic
Acid) solvent systems. The identification of highest polarity of compounds were shown to
elute farther from the origin point of activated silica gel using different type of eluents
(mobile phase) ratio.

Fig.1. Internal section of lactating rat’s abdominal part pretreated with JEC therapeutic
dosages.
Fig. 1.1. Internal section of abdominal stomach of lactating rats’ in 5th
day of JEC
treatment (500mg/kg/day & 1500mg/kg/day)
Notes; The arrow shows the intensity of yellowish coloration in rats’ inner abdominal
stomach based upon different JEC relative therapeutic dosages given.
Fig. 1.2 Internal section of abdominal stomach of lactating rat’s in 5th
day of JEC
treatment (150mg/kg/day)

Fig. 1.3 Internal section of abdominal stomach of lactating rat’s in 5th
day of JEC
treatment (0mg/kg/day)

Figure 2.1: Chromatogram profile of aqueous and organic fraction chromatogram of liver
extract ; 1500mg/kg/day of JEC relative therapeutic dosages using Chloroform : Acetic Acid
(90:10/100 %); (9:1, v/v) solvent system; under ultraviolet light (366nm) detection.
Reference:
1.Aqueous phase of liver extract (negative control)
2.Aqueous phase of liver ;1500mg/kg/day of JEC relative therapeutic dosage (7th days)
3.Aqueous phase of liver; 1500mg/kg/day of JEC relative therapeutic dosage (10th days)
4.Aqueous phase of liver ; 1500mg/kg/day of JEC relative therapeutic dosage (14th days)
5. Organic phase of liver extract (negative control)
6. Aqueous phase of liver ;1500mg/kg/day of JEC relative therapeutic dosage(7th days)
7 Aqueous phase of liver ;1500mg/kg/day of JEC relative therapeutic dosages (10thth days).
8. Aqueous phase of liver ;1500mg/kg/day of JEC relative therapeutic dosages (14th days)
9. Reference JEC (2.5mg)
Rf= 0.87
Rf= 0.75

Figure 2.2 Chromatogram profile of aquoues and organic fraction on liver extract;
1500mg/kg/day of JEC relative therapeutic dosages using Chloroform : Acetic Acid
(90:10/100%); (9:1, v/v) solvent system; under ultraviolet light (254nm) detection.
Reference:
1 .Aqueous phase of liver extract (negative control)
2.Aqueous phase of liver ;1500mg/kg/day of JEC relative therapeutic dosage (7th days)
3.Aqueous phase of liver ;1500mg/kg/day of JEC relative therapeutic dosage(10th days)
4. Aqueous phase of liver ;1500mg/kg/day of JEC relative therapeutic dosage (14th days)
5. Organic phase of liver extract (negative control)
6. Aqueous phase of liver ;1500mg/kg/day of JEC relative therapeutic dosage(7th days)
7 Aqueous phase of liver ;1500mg/kg/day of JEC relative therapeutic dosages (10thth days).
8.Aqueous phase of liver ;1500mg/kg/day of JEC relative therapeutic dosages (14th days)
9. Reference JEC (2.5mg)
The prominent fluorescence bands (yellowish green) shown eluted in Rf 0.86 or Rf 0.75 value
which representing the presence of hydrophilic flavonoid compounds (polar) absorbed in
liver and blood samples of the suckling neonates. The non- fluorescence compounds

particularly non polar compounds of flavonoid binding proteins can be identified as volatile
compounds which is easily to evaporate and diminished in ambient temperature (37 °C)
during sampling method. These conventional method seems tedious and only applicable for
qualitative screening methods of bioflavonoids.
Table 1.1 (a) Preliminary studies shown the lactating rat’s pretreated with relative
therapeutic dosages of JEC in 0mg/kg/day; 150mg/kg/day; 500mg/kg/day and
1500mg/kg/day and showing significant results as shown in table below for acute and
chronic studies (n samples=144);
List of
samples
Plasma Protein samples Therapeutic JEC dosages (Chronic
study)
JEC
Results:
(-ve;+ve)
1.(a) Liver
0mg/kg/day ;carboxymethyl selulose
(CMC) dissolved with saline water
-
1.(b) Milk -
1.(c) Plasma sample (Blood) -
2.(a) Liver
150mg/kg/day; JEC dissolved with water
+
2.(b) Milk +
2.(c) Plasma sample (Blood) +
3.(a) Liver
+
3.(b) Milk +
4. (a) Liver
1500mg/kg/day; JEC dissolved with
water
+
4.(b) Milk +
4. (c) Plasma sample (Blood) -
Notes; Results on tissue samples of plasma protein samples upon relative therapeutic of JEC
dosages treatment. (+ve); positive results showing presence of JEC absorbed components; (-
ve); negative results showing absence of JEC absorbed components)

List of
samples
Plasma protein samples Therapeutic JEC dosages (Acute study)
JEC
Results;
(-ve;+ve)
1.(a) Liver
0mg/kg/day ;
carboxymethyl selulose (CMC) dissolved
with saline water
-
1.(b) Milk -
2.(a) Liver
+
2.(b) Milk +
3.(a) Liver
+
3.(b) Milk +
3. (c) Plasma sample (Blood) +
4. (a) Liver
+
4. (b) Milk +
4. (c) Plasma sample (Blood) +
Notes; Results on tissue samples of plasma protein samples upon JEC relative therapeutic

Table 2.1 (b) Preliminary studies, qualitative methods shown on suckling neonates
pretreated with relative therapeutic of JEC dosages in 0mg/kg/day; 150mg/kg/day;
500mg/kg/day and 1500mg/kg/day and showing significant results as shown in table below
for acute and chronic studies (n samples =144);
List of
samples.
Plasma protein’s sample Therapeutic JEC dosages (Chronic study) JEC
Results;
(-ve/+ve)
1.(a) Liver
0mg/kg/day ;carboxymethyl selulose
-
1.(b) Milk -
1.(d) Blood Brain Barrier -
2.(a) Liver
+
2.(b) Milk +
2.(d) Blood Brain Barrier +
3.(a) Liver
+
3.(b) Milk +
4. (a) Liver
+
4.(b) Milk +
Results on tissue samples of plasma protein samples upon JEC relative therapeutic dosages
treatment. (+ve); positive results showing presence of JEC absorbed components; (-ve);
negative results showing absence of JEC absorbed components)

List of
samples
Plasma protein’s sample Therapeutic JEC dosages (Acute study)
JEC
Results;
(-ve/+ve)
1.(a) Liver
0mg/kg/day ; carboxymethyl selulose
-
1.(b) Milk -
1.(d) Blood Brain Barrier -
2.(a) Liver
+
2.(b) Milk +
3.(a) Liver
+
3.(b) Milk +
4. (a) Liver
+
4.(b) Milk +
4. (d) Blood Brain Barrier +
Notes; Results on tissue samples of plasma protein samples upon JEC relative therapeutic
The optimization of HPLC and LCMS analysis were developed and showing abundance of
flavonoid binding protein plasma derived from suckling neonates’ tissues (blood brain barrier
tissues) and liver. The presence of abundance unique peaks in HPLC (High Performance
Liquid Chromatography) analysis and high throughout LCMS (Liquid Chromatography Mass
Spectrometry) based on eluted retention time (Rt), within time frame of analysis giving the
good insight in xenobiotic metabolism (biotransformation of xenobiotic) that simply occur in

the liver. Liver (hepatic samples) known for biotransformation of xenobiotic metabolism
occurrence whereby the foreign compounds begins to interact with mix function oxidase
enzymes in phase 1 and converting the xenobiotic into hydrophilic and rendered to be
eliminated out (Administration; Distribution; Metabolism and Excretion) throughout the
body. The metabolism of xenobiotics, perhaps the most notable pathway is the
monooxygenation function catalyzed by the cytochrome P450s (CYPs; P450s). The CYPs
detoxify and or bioactivate a vast number of xenobiotic chemicals and conduct
functionalization reactions that include N- and O dealkylation, aliphatic and aromatic
hydroxylation, N- and S oxidation, and deamination[6].
The vulnerability and poor development of neonate’s itself, enabling the permeability
and susceptibility towards the drug absorption in liver and transverse into the blood brain
barrier even at lower dosage of relative therapeutic JEC. The results shown the fluorescence
bands emanated from JEC components; nursed by dams even pre-treated at lowest dosages of
JEC (150mg/kg/day) in which undergone biotransformation process in phase 1 liver into
hydrophilic compounds, which rendered to be passively diffused out from liver to the hepar
portal vein before finds it route to transverse blood brain brain in suckling neonates. Previous
study shown the JEC treatment induce the GABA α- receptor that is mediating the sedative
effects; ptosis, anxiolytic effect in mice that shown in drug agonist GABA receptor such as
imidazole and benzodiazepine. The bioaccumulation of JEC compounds in liver and brain
prior to long term of drug exposure even in lowest dosages (150mg/kg/day) may cause
adverse effect or simply said exhibit the toxicity effects towards the neonates at early stage of
development particularly in cognitive impairment and growth development (body weight),
metabolic functions and physiological behaviour in suckling neonates.
The peak absorbance shown upon HPLC and high throughput sensitive LCMS
analysis showing abundance of flavonoids compounds based on retention times eluted out
using standardize mobile phase. The higher molecular weight metabolite compounds will
elute farther within time frame of analysis based on resulted Rt and eluted time shown.
Kaempferol, one of flavonoid compound were detected to be appeared in most all the
subjected tissues samples (liver of maternally ingested JEC extract; brain and blood samples

of suckling neonates) persistent with therapeutic dosages given even in highest dosages of
therapeutic JEC dosages (1500mg/kg/day). One of the peak formed was identified similar to
bioflavonoid compound, kaempferol quantified as 0.57mg, partition only 1.1348% in the
sample (n=3) and having turnover 0.38% from maternal ingested dosages (150mg/kg/day).
Notably, this peak has similar properties of molecular mass (287.0561 and 449.1094 max.
m/z) at Rt (24.9 and 30.1 min) with JEC and kaempferol standard profile. These findings
significantly shown the JEC components are able to be cleared rapidly in the pre-hepatic
circulation within short time of post-drug and the other metabolite products of the drugs
which undergone biotransformation in hepar could be traced inside the brains of neonates
nursed by dams even in the lowest dosage. These subjected plasma tissues sample were
quantitatively tested with incorporation of spiked kaempferol as internal standard in plasma
binding protein brain tissues of suckling neonates compared to kaempferol standard, and
plasma tissues of control treatment and showing the presence of kaempferol, In addition,
LCMS chromatogram profiles of maternal ingested JEC of milk tissues, discerning of higher
yield of unique peaks resulted in comparison with the suckling neonates tissues and absence
in for both control samples.

Fig 3.1 LCMS chromatogram profile of aqueous phase of neonate’s liver extract
nursed by dam pretreated with lowest; 150mg/kg/day relative therapeutic JEC dosages in
chronic studies (5th
of post-drug). Peaks shown were identified by comparison with reference
standards on retention time. The profile clearly shown presence of bioflavonoid (bioactive
compounds) demonstrated on distinctive number of peaks yield, and peaks no. 15.; identified
as kaempferol (26.7 min); ion m/z 285.2889 , and compared to kaempferol standard whilst
absence in control sample within elution time (RT) 30 min.

Fig. 3.2 LCMS Chromatogram profile , mass spectra of aqueous phase of neonate’s
liver extract nursed by dam pretreated with lowest; 150mg/kg/day relative therapeutic JEC
dosages.

Fig. 3.3 LCMS chromatogram profile on kaempferol as reference standard (1.0µg/ml)
to provide the best resolution comparison with tissues plasma protein sample pretreated JEC;
150mg/kg/day and 0mg/kg/day; control sample.
Conclusions
These useful informations, thus clearly implying that there is abundance of bioactive JEC
plant derived compounds known as secondary metabolite bioflavonoids or (origin from
parent compound); such as quercetin, quercitrin and astragalin as such which having
pharmacological properties, such as antioxidant agents that being able to adhere and retain in
protein sample tissues even in lower dosages of JEC treatment. This clearly shows the use of
this plant as herbal remedies to evoke the understanding on pharmacology and
pharmacokinetic of therapeutic dosages of drug intake in dietary consumptions which is dose
dependent versus time of drug exposure. Despite all the challenges in producing informative
data, these minimal findings provides good insight and useful information in optimizing the
characterization of bioflavonoids, phenolic acid which is JEC absorbed compounds in plasma
tissues via modern, high resolution and reproducibility analysis using NMR techniques in
providing productive data bank of biosynthesis flavonoids compounds.

Acknowledgements
The author are thankful to Biochemistry department’s staffs, lecturers, Food Science and
Technology’s Department staff, Food and Chemistry’s Department staff and Animal House’s
staff in Faculty of Science and Technology, National University of Malaysia, UKM, Bangi,
Selangor, Malaysia. These perpetual research has been funded by FRGS grants in aiding the
instrumentation research analysis and chemicals.
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Characterization of jec absorbed compounds in lactating rat's milk and suckling neonates

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