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2.a OPTIMIZATION CHARACTERIZATION OF JEC ABSORBED COMPOUNDS IN LACTATING RATS AND SUCKLING NEONATES NEW

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2.a OPTIMIZATION CHARACTERIZATION OF JEC ABSORBED COMPOUNDS IN LACTATING RATS AND SUCKLING NEONATES NEW

  1. 1. 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.
  2. 2. 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).
  3. 3. 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
  4. 4. 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.
  5. 5. 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-
  6. 6. 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.
  7. 7. 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.
  8. 8. 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)
  9. 9. Fig. 1.3 Internal section of abdominal stomach of lactating rat’s in 5th day of JEC treatment (0mg/kg/day)
  10. 10. 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
  11. 11. 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
  12. 12. 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)
  13. 13. 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)
  14. 14. 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)
  15. 15. 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
  16. 16. 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
  17. 17. 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.
  18. 18. 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.
  19. 19. 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.
  20. 20. 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.
  21. 21. 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. References; [1] Allen Y. C and Yi C.C. 2013. A review of the dietary flavonoid, kaempferol on human health and cancer chemoprevention. J. Food Chemistry.138. 2099-2107. [2] A. Hasler, O. Stichler. 1990. Analysis of quercetin and kaempferol in Gingko extract and tablets (Gingko Bilboba) by HPLC. J. Chromatogr. 508. 236-240. [3] Baldwin, M.A., Carter, D.M., Darwish, F. A, and Phillipsonm, J.S., (1981), Biomed. Mass Spectrum, B., 362. [4] Darwish FA, Evans JF, Phillipson J.D. (1979), Cytotoxic bruceolides from Brucea javanica. J Pharm Pharmacol (suppl) 31:10. [5] Casteele, K. V., Geiger, H., and Sumere, C.F., 1981. Chromatography. Separation of Flavonoids by reverse-phase high performance liquid chromatography. Elsevier. Amsterdam. [6] Curtis. J.O, John P., Vanden H., Gary H. P., and Jeffrey M. P. 2011. Xenobiotic Metabolism, Disposition, and Regulation by Receptors: From Biochemical Phenomenon to Predictors of Major Toxicities., Toxicological Sciences 120(S1), S49–S75. [7] Cotton, C.M., 1996. Ethnobotany: Principle and Application. John Wiley and Sons, New York. [8] D. Strack, V. Wray, 1989. Anthocyanins in Methods, Plant Biochem. 325-326. [9] E. Middleton, Jr. and C. Kandaswami,1994.The Flavonoids, Advances in Research since 1986’ (J.B. Harborne, ed.) p. 619. Chapman and Hall, London. [10] Earnsworth, N.R., Berderka, J.P. and Moses, M. 1974. Screening of Medicinal Plants. Journal of Pharmaceutical Science 63: 457-459. [11] F.M. Wang, T.W. Yao, S. Zeng, 2003. Determination of quercetin and kaempferol in human urine after orally administrated tablet of ginkgo biloba extract by HPLC. J. Pharma. Biomed. Anal. 33. 317-321. [12] Harbone,J. M. and Baxter, H. 1993. Phytochemical Dictionary: A handbook of bioactive compounds from plants. Taylor and Francis Ltd. London. Washington DC, USA. 755pp. [13] Ian R. Phillips and Elizabeth A. Shepard., 2006. Methods in Molecular bio Volume320, CYP450 Protocols Second Edition. Humana express.
  22. 22. [14] Lincoln, D. W., Hill. A. & Wakerly, J.B. 1973. The milk ejection reflex of the rat: an intermittent function not abolished by surgical levels of anaesthesia. J. Endocr. 57, 459-476. [15] Lincoln , D.W. & Wakerley, J. B. 1975. Factors governing the periodic activation of supraoptic and paraventricular neurosecretory cells during suckling in the rat. J. Physiol. 250, 443-461. [16] Mst. A. K., Md .H. R., Mohammed R. 2011. Scientific Validation of Eight Medicinal Plants Used in Traditional Medicinal Systems of Malaysia: a Review. American- Eurasians.5 (1); 67-75. [17] Marianna R. and Magdalena R. 2015. Analysis of plant lipids. Plant Lipids Science, Technology, Nutritional Value and Benefits to Human Health: 221-238. [18] Markham, K. R., and T.J. Mabry: Phytochemistry 7, 791(1968). [19] Obouayeba A. P., Djyh N. B., Diabete S., Djaman A. J, N'Guessan J. D., Kone M., and Kouakou T. 2014. H., Phytochemical and Antioxidant Activity of Roselle (Hibiscus Sabdariffa L.). Research Journal of Pharmaceutical, Biological and Chemical Sciences. 1453-1465. [20] Paul E. M., and Wilhelmina K. 2010. Xenobiotic Metabolism and Berry Flavonoid Transport across the Blood−Brain Barrier. J. Agric. Food Chem .58(7), pp 3950– 3956. [21] Parke, D. V. 1984. The biochemistry of foreign compounds. New York: PergamonPress. [22] P. M. D. and Harbone. J.B, 1989. Handbook of Methods in Plant Biochemistry. Plant Phenolics. Volume 1: 1-23 [23] Q. Zhang, Y. Zhang, Z. Zhang, Z. Lu. 2009. Sensitive determination of kaempferol in rat plasma by high-performance liquid chromatography with chemiluminescence detection and application to a pharmacokinetic study. J. Chromatogr. B 877.3595- 3600. [24] Seikel, M. K., in: The chemistry of flavonoid compounds (edited by T.A. Geissmen),p. 34-69. Oxford, Pergamon Press. 1962. [25] S.P. Jun, H.S. Rho, H.K. Duck, I.S. Chang. 2006. Enzymatic preparation of kaempferol from green tea seed and its antioxidant activity, J. Agric. Food Chem. 54. 2951-2956. [26] Srovnalova A. , Svecarova M. , Zapletalova M. K, Anzenbacher P, Bachleda P, A., E, Dvorak Z., 2014. Effects of anthocyanidins and anthocyanins on the expression and catalytic activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in primary human hepatocytes and human liver microsomes. J Agric Food Chem. Jan 22; 62(3):789-97. [27] “The Flavonoids, Advances in Research since 1986” (J.B. Harborne, ed).. Chapman and Hall, London, 1994. [28] Wagner and Bladt. 1996.Plant Drug Analysis: A thin layer chromatography Atlas, (2nd Edn.)Springer-Verlag, Berlin Heidelberg London: New York. 349–354. [29] Wakerley. J. B. &, O. Neill, D.S. & Ter Haar, M. B. (1978). Relationship between the suckling-induced release of oxytocin and prolactin in the urethane-anaesthetized lactating rat. J. Endocr. 76, 493-500. [30] WHO, 2003. Traditional Medicine , WHO, Geneva. Website; http://www.cyberlipid.org/extract/extr0010.htm (Soxhlet France Ann, 1879).

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