PALM DATE KERNELS AS UNCONVENTIONAL FUNCTIONAL FOOD INGREDIENT OF NATURAL PHYTOESTROGEN IMPROVING HEALTH OF MENOPAUSES FEMALE RATS

PALM DATE KERNELS AS UNCONVENTIONAL FUNCTIONAL FOOD INGREDIENT OF NATURAL PHYTOESTROGEN   IMPROVING HEALTH OF MENOPAUSES FEMALE RATS

Mohamed M.  El-Sayed Ali, Tarek Abd El-Rahman  and Asmaa Moath.

Faculty of  Home  Economics, Minufiya University,  Egypt.

ABESTRACT

  This study was conducted to fined out probable benefit of palm date kernels phoenix dactylifera  representing natural estrogen since for  menopausal rats( 205± 2g each) .Kernel, of two varieties of ripe palm date were used, which were Amhat and Zaghlool .

Chemical analysis revealed that kernels were rich in fibers (69-70%).Powdered kernels added at 5 and 10% levels to diet . levels of Hb RBC's, WBC's  ,NEU, MONO, EOS,BASO(white blood cells), was determined in blood ,while  in serum analyses included; liver and kidneys functions indices is GPT,GOT,ALK.P, ALP, T.B, creatinine ; Cholesterol and glucose were also assessed. Estrogen level was determined and femur (right & left) density measured. The results indicated the value of palm date kernels for  health of menopause female rats.

        Keywords: Palm date kernels, unconventional functional food, natural phytoestrogen,   health menopauses, female rats.

INTRODUCTION

Menopause is a natural process that happens to every woman as she grows older, and it is not a medical problem, disease or illness, still some women may have a hard time because of the changes in hormone levels during menopause (Anon, 2006).The women going through induced menopause may have more several menopausal symptoms, and are usually treated with hormone therapy which had potential side effects, this is in medicine. It emerged from ranking tasks that functional foods do not constitute a product category as such; they occupy an anomalous position between "Food" on one hand and . Medicine on the other participants tend to dislike the idea of a" personal" food not intended to comprise part of a shared meal in the household, and also tend to distrust  health claims promoted by the food industry. The study of Korzen and O'Doherty, (2006) indicates a need for further research in these nutrition areas. to face these symptoms and physiological action in these stages of age.  Several foods could face side by side drugs or without, that have a natural hormones called phytoestrogen like in black cohosh, soya, whole grains, flaxseed, banana fruit beside the role of several nourish in nutrients. To treat a lot of symptoms like Mn, Si, B₁₂, and E found in vegetables and fruits which we usually took in ordinary diet. Not only ordinary diet used to face menopause symptoms but also unconventional diet does this which the present work discussed. unconventional diet that may be  seeds from ordinary diet. So, the seeds or kernels of date palm come to light. as one of unconventional diet with phytoestrogen in their  contents. Therefore, the present study is work was carried out to investigate the effect of that unconventional diet on menopauses female rats health improving.

SUBJECTS AND METHODS

I.1.Palm Date kernel's preparation.

          Palm date phoenix dactyliferaAmhat and Zaghlool kinds were ripe and purchased from Egyptian local markets. Kernels were separation from palm date fruit, washed with distilled water, cleaned   then dried at 60C^°contenuosly for to two week's in dryer, grilled then milled.

2. Experimental Animals.

          Female menopause 40 rats (Sprague Dawley strain) weighting 205± 2 were used in this investigation. The animals were obtained from Research Institute of Ophthalmology, Giza, Egypt. Each rat was housed in special cage under controlled condition. Every day the animals were observed for the external appearance, shape, color, distribution of hair and physical activity. All rats were fed for 7 days on the control diet before the beginning of the experiment. The rats were weighed after 3 days (each separately). The diet was presented to rats in special covered cups to avoid food loss. All rats were provided with water by glass tubes through wire cage. The rats were fed a diet ad libitum throughout the period of the experiment, after that weighed once week .Rats was kept on experimental diet for seven weeks. They were classified to four major groups, which had four rats each vs. control group, according to the following.

Table (a): Classification of experimental groups.

I.3.Basal diet

       Basal diet and salts mixture was prepared according Hegested et al., (1941). While, vitamin mixture according to Campbell (1963).

1. Blood samples.

 Blood sampling:

            At the end of the experiment, rats were fasted over night and anesthetized with chloroform. Blood samples were collected in clean dry centrifuge tubes from hepatic portal vein. Blood was centrifuged for 10 minutes at 3000 rpm to separate the serum, which were kept in tubes at – 18°C till analysis.

III. Separation of organs:

     Rats were subjected ether vapors inside desiccators for few second to be narcotized according to Organs were separated at the end of the experiment by excising the animals with surgery then transported by forceps Organs were taken, washed with saline and dried with filter paper, then weighed and kept in neutral formalin solution. and put into Bouin solution for histological studies (fixation).

IV .Biochemical analysis:

IV.1-Determination of moisture and ash:

      Moisture, protein content, fat, fibers and ash contents were determined according to methods described by the A.O.A.C. (1994). Carbohydrate was estimated by difference as follows: carbohydrate = 100- (% protein + % fat + % moisture +fibers + % ash).

IV .2. Hematological and serological assay.

            Enzymatic colorimetric method used to determine blood glucose according to Trinder (1969). Read blood cells, White blood cells and hemoglobin determined according to Dacie. and Lewis (1991). Determination of cholesterol was according to Anon (1987). Colorimetric method used to determine AST according to Anon,(1980). Concerning ALT it was determined according to Hfkenscheied (1979). Kinetic determination of alkaline phosphatase (ALP) activity was according to Belfied and  Goldberg,, (1971) , plasma total protein according to Weichselbaim (1964), plasma albumin according to Doumas and Biggs (1971). Colorimetric method was used to determine creatinine according to Bohme,, (1971) estrogen (E₂) was detrmined according ANON(1990). Bone density was determiend according to El-Adawy (1986).

IV.3. Statistical Analysis

Statistical analysis were performed by using computer program statistical package for social science (SPSS, 1998), and values compared with each other using the suitable tests.

V. Histopathological studies.

        The obtained organs were fixed by using of Bouin’s solution according to the method of Humanso (1962). The aqueous Bouin’s solution was prepared as follow: Saturated aqueous solution of picric acid, formalin 25% Cm3, acetic acid 5 cm. Microscopic sections from organs prepared according to the method of Kiernan (1981).

VI. C.T ray diagnosis:

   C.T diagnosis and photographic was done by technical and support of staff of Department of Radiology Research Institute of Ophthalmology, Giza, Egypt.

RESULTS AND DISCUSSION

Table 1 results show the proximate composition of palm 'date kernels. It could be observed that carbohydrates and fiber are the major components for two date seeds types. 70.22%and69.2% are the contents of carbohydrates, and 12,11 are the percents of fiber for ripe Zaghlool and Amhat date seeds, respectively: These values considering the chemical composition fall within the range quoted by other investigators (Abd El'Nabey, 1999, Shrud et al .,2001 and Awadalla et al., 2002).

Table 1:  Proximate composition of date seeds

From results of Table 2 is evident that Hb, RBC's, WBC's& NEU increased by feeding on date kernel diets, while MONO & EOS decreased .In general these changes indicate better heal health of rats.

             Data in Table 3 show the effect of palm date kernels on some serum liver function parameters. Alkaline phosphates (u/l) results being significant difference for group 5 if it is scored at high value of ALP P=0.015, 102.5±0.28 (Mean± SD). But for group 3 scored the lowest value of ALP (p=0.001) 91.75±0.25 (Mean ± SD), the highest ALP, however was found for 5% Amhat date seeds diet (group 2) (107.7±0.25 u/l) and difference was high significance compared to control (-) group.  the greatest concentration of ALP are found in bone and liver (where proliferation of epithelial liming the bile duct and focal area  of  hepatic  hemorrhage  found  in  histopathological  examination. Partly

             Table 2 :  Effect of palm date kernels on complete blood count CBC).

P<0.05* Significant,  P<0.01 **High significant , P<0.001*** Very*high significant

Table 3 :  Effect of palm date kernels on some liver functions.

P<0.05* Significant , P<0.01** High significant,            P<0.001*** Very high significant

every body tissue contains at least a small amount of ALP.  Because of this wide distribution, limited information can be obtained from a total ALP assay elevated serum and leukocyte ALP levels in patients with Hodgkin's and non-Hodgkin's lymphoma were reported by several investigators(Thysset al.,1985). Glutamate oxaloacetate trans aminase (GOT) was detriment result (u/l) obviously there is no significant differences for all studied groups after treatment with palm date kernels except for 10% Amhat and 10% Zaghlool diets. But the highest value was for group 3,it was 66±0.25 (Mean±SD) after control one 58.5±0.28 (Mean ± SD). GOT is also an indicator for healthy liver .It means there is no side effect in liver rat when they treated with palm date kernels in basal diet. Results related glutamate pyruvate trans aminase (GPT) revealed insignificant difference, except only for group 3 achieved (p<0.05). 53.7±0.25 (Mean ± SD) Insignificant difference recorded for all other groups. GPT is an indicator for healthy liver, but for group 3 there is an increase in GPT according to hepatic sinusoids and necrosis of sporadic hepatocytes, as Abd El Aziz (1990)published that value of GOT and GPT are elevated in early all liver diseases but are particulary high in conditions that causes  extensive cell necrois, such as severe viral hepatitis and prolongated circulatory collapse. Serial enzyme measurements are often useful in determining the course of liver damage, but it is not sick case if all serum liver function parameters is normal (up to 45 u/ml for GPT). Serum albumin  values showed that highest level was scored for group 2 and group 3 (p>0.05-0.01), 4.17±2.5(Mean±SD) which fed on Amhat date  seeds in their basal diet. On the other side, group 4 and group 5 had the lowest value (p>0.05) 3.7±7.07,4.12±2.5 (Mean±SD) respectively which are fed on  Zaghlool date seeds in their basal diet. Albumin is one of plasma  and proteins. It is synthesized in the liver, 50% are synthesized in the liver 50% are synthesized in the plasma cells of reticuloendothelial system (RES) which is  diffuse system of cells present in the liver, spleen, lymph nodes and bone marrow. Albumin act as a carrier for substances as hormones.vitamins ,lipids and minerals to prevent their loss in urine. Albumin decreased in 1) liver diseases, as liver cirrhosis and infective hepatitis, since the liver does not produce sufficient albumin 2) kidney diseases, e.g. nephrosis as the albumin with its small molecular size is lost in urine (Hassan, 2005). All difference of albumin were in normal range  indicating ,it showed healthy kidney and liver although simple necrosis in liver tissue which histopathological examination result .

Total protein (T.P) determination revealed highest value for group 3, then group 2 (P>0.01<0.05) 7.07±2.5, 6.7±2.88 (Mean ± SD) respectively but the lowest value for the other groups. Plasma proteins are albumin, fibrinogen and prothrombin; they are synthesized in the liver. Globulins( 50%) are synthesized in the in the liver. 50% are synthesized in the plasma cells of the reticuloendothelial system (RES) which is a diffused system of cells present in the liver, spleen, lymph nodes and bone marrow. Its concentration was (7.2:7.4mg/dl) (Hassan, 2005). distinct order of changes .There is no effect on total serum proteins due to date kernels and it is an indicator for good liver that synthesized 50% of them.

 Results in Table 4 illustrated the effect of different treatments with palm date kernels on serum glucose (mg/dl) of rats for all studied groups. It could be observed:  Significant difference was found for group 3 and group 5,

Table 4 : Effect of palm date kernels on sugar and creatinine serum

P<0.05* Significant, P<0.01**high significant, P<0.001 *** Very high significant.

where rats fed on the highest level of palm date kernels (P=0.006, 1<0.05), 116.75±0.28, 116.75±0.25 (Mean ± SD), respectively. There is non significant difference for the other groups 2, 4 if (p=0.0) (123.5±0.28), (113.75±-0.25), (Mean+SD) when compared with control group. High fiber and phytoestrogen content for flaxseed leads to as affect as oral estrogen-progesterone to improve mild menopausal symptoms and to lower glucose and insulin levels (Lemay et al., 2002), the same could be observed for palm date kernels (groups 3-5).Possibly high fiber diet for group5 (10% Zaghlol date seeds diet) and group 3 (10% Amhat date seeds diet) respectively my be for reductions serum glucose.

Data concerned with effect of different treatment with palm date kernels on serum creatinine are shown in Table 3. Was significant differences could be noticed for all studied groups in comparison with control. Creatinine is an indicator for healthy kidney. It is normal for all studied groups (0.5:0.6) mg/dl .So, it could be concluded that there were no side effects on kidneys after feeding of rats with palm date kernels in rats diet.

Data of Table 5 show the effect of feeding with two types of palm date kernels on serum cholesterol of rats. It could be noticed the cleared nosignificant differences for all studied groups ,values were the normal level of serum cholesterol (<200mg/100ml) ,but the highest value scored for group 5 in T.test 192.5 ±0.5 (Mean ± SD). As cholesterol was  in normal value, palm date kernels succeded in being  a source of phytoestrogen to improve  and ameliorate menopausal symptoms without hypercholesterolemia, therefore it could disagree with what was published "only hormone replacement therapy significantly improve cholesterol profile in hypercholesterolemic women, and favorably modifies markers related to cardovascular" (Lemay et al.,2002). Also high fiber content 15% of palm date kernels helped to maintain cholesterol in normal ratio (Anon,  2006).

Results in Table 5 show the effect of treatment with palm date kernels on serum estrogen (E2)of rats for all studied groups. It could be observed  compared to control (-) significant difference was found for group 5, that scored the highest value (p<0.05) which was  78.5±0.28 (Mean±SD) .Non significant difference for other groups recorded provided that  they all  were in normal ratio . There is a relationship for energy, fat and fiber intakes from in palm date kernels to plasma concentrations of estrogens and androgens in premenopausal women (Rose et al., 1991). Results of this cross sectional study support a relation between both energy and fat ingestion and plasma sex hormone concentration in premenopausal women. Palm date kernels are high in β- sitosterol and stigmasterol which helped in estrogen synthesis (Abd El Wahab et al., 1997). Although that last relations, leading to increase in serum Es did not observed. As estrogens are produced from pre follicles in amounts that increase gradually before ovaration directly to reach the highest value before ovaration, and produced on three forms, which are oestradiol, oestrone and oestriol. They are found in blood circulation and secreted in bile and urine combined with acids like glucoronic acid, that combination induced in liver after arriving the highest value before ovaration and produced on three forms, which are oestradiol, oestrone and oestriol..LSH and FSH were stimulated from hypothalamus for ovaration (Rashad and Khalil, 1998). Oestradiol concentration in the serum of female rats decreased significantly as the present of date pits increased, this may be due to the estrogenic effect of date pits (Aldhaheri et al., 2004), it is the reaction which phytoestrogen in palm date kernels do actually and histopathological examination of ovaries revealed that. Also linolenic fatty acid that palm date kernels contain has an effect on ovary Ponter et al. ( 2006) published that the increased linolenic supply to the cows had only a modest effect on follicle populations and corpus luteum activity was unchanged.

Table 5 : Effect of palm date kernels on cholesterol and estrogen.

Data listed in Table 6 and Figure 1 show the effect of different treatments with palm date kernels on femur bone density of rats for all studied groups. It could be noticed that there is non significant difference for all groups  in comparison  with control.  All values  were in normal  level.  Anon  (2006) reported also that body weight is a major predictor of forearm bone mineral density (BMD); among the factors measured in this study on independent Japanese women, 69 years of age and over serum concentration may be associated with cortical BMD Maintenance of body weight is very important to maintains BMD in this population unless a large weight aggravates obesity related diseases. a nevertheless follow up study is needed to confirm these findings (Nakamura et al., 2006).

Table 6.  Showed the effect of date palm kernels on femur density

  Group1

  Group2 

     Group 3

     Group  4 & Group 5

                     Figure 1.  Femur view due to feeding treatments.

Histopathological studies:

Examination of uterus.

Examination of ovaries:

Conclusively, This study was conducted to fined out probable benefit of palm date kernels phoenix dactylifera representing natural estrogen since for menopausal rats. The results indicated the value of palm date kernels for health of menopause female rats.

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نواة تمر النخیل کغذاء وظیفى غیر شائع محتوى على استیروجین نباتى طبیعیا یحسن صحة اناث الفئران المتوقفه عن الأنجاب

محمد مصطفى السید على ، طارق عبد الرحمن ، اسماء معاذ

قسم التغذیة- کلیة الأقتصاد المنزلى- جامعة المنوفیه 

       اهتمت هذه الدراسه بامکانیة الأستفاده من نوى تمر النخیل phoenix  dactylifera   الذى یحتوى على ایستروجین طبیعیا علىاﻹناث المتوقفه عن الأنجاب( 205±2جم لکلن). واستخدم نوعین من نوى تمر النخیل لثمار ناضجه،وهى الأمهات والزغلول.واوضح التحلیل الکیمیائى غنى  النوى من الألیاف (69-70%) . اضیف مسحوق النوى بنسب 5و 10% الى العلیقه،  وتم قیاس کلا من  HbوRBC's وکذلک   WBC's NEU, MONO, EOS,BASO( کرات الدم البیضاء)فى الدم ، بینما قدر فى المصل دلالات وظائف الکبد والکلیتین وهى GPT,GOT,ALK.P, ALP, T.B, creatinine کما قدر الکولیسترول والجلوکوز. قدر مستوى الأستروجین وکثافة عظم الفخذ ( الأیمن والأیسر). اوضحت النتائج قیمة نوى تمر النخیل لصحة اناث الفئران المتوقفه عن اﻹنجاب.

التوصیة: امکانیة الأستفاده من نوى تمر النخیل phoenix  dactylifera    الذى یحتوى على ایستروجین طبیعیا علىاﻹناث المتوقفه عن الأنجاب , اوضحت النتائج قیمة نوى تمر النخیل لصحة اناث الفئران المتوقفه عن اﻹنجاب.