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

500mg/kg/day; JEC dissolved with water

+

3.(b) Milk +

3.(c) Plasma sample (Blood) -

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 -

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

500mg/kg/day; JEC dissolved with water

+

3.(b) Milk +

3. (c) Plasma sample (Blood) +

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 JEC relative therapeutic

dosages treatment. (+ve); positive results showing presence of JEC absorbed components; (-

ve); negative results showing absence of JEC absorbed components)

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

(CMC) dissolved with saline water

-

1.(b) Milk -

1.(c) Plasma sample (Blood) -

1.(d) Blood Brain Barrier -

2.(a) Liver

150mg/kg/day; JEC dissolved with water

+

2.(b) Milk +

2.(c) Plasma sample (Blood) +

2.(d) Blood Brain Barrier +

3.(a) Liver

500mg/kg/day; JEC dissolved with water

+

3.(b) Milk +

3.(c) Plasma sample (Blood) -

3.(d) Blood Brain Barrier +

4. (a) Liver

1500mg/kg/day; JEC dissolved with water

+

4.(b) Milk +

4.(c) Plasma sample (Blood) -

4.(d) Blood Brain Barrier +

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

(CMC) dissolved with saline water

-

1.(b) Milk -

1.(c) Plasma sample (Blood) -

1.(d) Blood Brain Barrier -

2.(a) Liver

150mg/kg/day; JEC dissolved with water

+

2.(b) Milk +

2.(c) Plasma sample (Blood) +

2.(d) Blood Brain Barrier +

3.(a) Liver

500mg/kg/day; JEC dissolved with water

+

3.(b) Milk +

3.(c) Plasma sample (Blood) +

3.(d) Blood Brain Barrier +

4. (a) Liver

1500mg/kg/day; JEC dissolved with water

+

4.(b) Milk +

4.(c) Plasma sample (Blood) +

4. (d) Blood Brain Barrier +

Notes; 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)

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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