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ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Preliminary Study to Determine the Effects of Chemical solutions (Mahalabiah& Serratia) Added to the on Biochemical and Hematological Parameters in white rabbits

Maryam Mohamed Besher*, Aisha abokhzam, Rajaa Alghani. Medical laboratory Sciences Department, Faculty of Engineering& Technology, Sabha University/Libya.

Received: July 2016, Accepted: September 2016 ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Abstract The objective of the study was to determine the Influence of some chemical additives to Henna and most actively traded on some liver and kidney functions and complete blood count in white rabbits.15 rabbit divided into three groups, each group containing five rabbits. The first group which used as a control was a topical application of henna on the dorsal region of the rabbits after removing hair. The second group had been used henna with Mahlbaih. Henna was used with Serratia in third group. Rabbits were slaughter of after 24 hours to get the blood to require analyzes. The results of this study showed a significant increase in AST, ALT and increase in the concentration of Urea and Creatinine in the two groups, which used Mahalbiah and Serratia. There is no variability in CBC count between three groups.

Key Words: Henna, Liver, Kidney, Blood, Rabbits, Chemical additives. ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ

Introduction: Scientific Classification of henna plant [5, 6, 7]: Henna or hina (Lawsonia inermis, family • Subkingdom: Tracheobionta Lythraceae) is a flowering plant or shrub native • Superdivision: Spermatophyta to tropical and subtropical regions of Africa, • Division: Magnoliophyta and Southern Asia. Preferred Scientific Name: • Class: Magnoliopsida Lawsonia Inermis. Other Scientific Names: • Subclass: Rosidae Lawsonia alba Lam. Trade Name: Henna. Most • Order: Myrtales Popular Common Names:Arabic; Henna, • Family: Lythraceae Henne, Hine, Hina, Hene, Heni. , • Genus: Lawsonia L & ; Mendhi, Mehndi, Mehendi [1, 2, • Species: Lawsonia Inermis L 3, 4]. Henna is commercially cultivated in Morocco, Libya, Sudan, India, Pakistan, Yemen, and other ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ *Corresponding author: Maryam Mohamed Besher Email: [email protected] countries [8-11], It contains a burgundy dye

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molecule, lawsone (2-hydroxy-1,4- Reports show that methanolic root extracts of naphthoquinone). This dye molecule has the Lawsonia is used in Nigeria for cosmetic ability to bond with proteins, and consequently purposes, as antimalarial as well as for has been widely used in body art to dye skin, abortifacient purposes. The powdered roasted hair and fingernails, and to dye silk, leather, and seed is mixed with gingerly oil to make a wool. Henna body art is made by applying paste which is used for the treatment of ring henna paste to the skin. Henna paste is prepared worm [18]. Decoction of the leaves is used by drying the henna leaves and grinding them to for aseptic cleaning of wounds and healing powder, and then this powder is mixed with oil [15, 16]. L. inermis is also used by some or water to form the paste. When this henna individuals as ‘blood tonic’, thus implying its paste is applied to the skin the dye (lawsone) multifaceted use [17]. migrates from the paste to the outermost layer Women of all ages use henna for skin of the skin; more lawsone will migrate if the decoration, and it is considered an essential paste is left on the skin for a longer time, thus part of the ceremonies and other creating a red-brown stain [12-15]. social celebrations. Despite the wide spread In the Arab world and Indian subcontinent use of natural henna, reports of allergic henna was used for skin decoration and hair contact dermatitis to natural henna and dying during social celebrations, and during influences of some solutions added to henna marriage ceremonies. People celebrate by are very rare in the literature [17]. adorning the bride, and sometimes the groom with henna [16]. Due to the frequent use of henna plant on the Lawsonia inermis Linn. (Lythraceae) is a skin and hair among women in the general very useful medicinal plant in all parts of the Arab societies, Libyan community in world [12, 13, 17]. The leaf powder of henna particular, frequent addition of chemicals sap was used for staining hair, nails and unknown origin and unknown composition of beard. The leaves of Lawsonia inermis are henna paste to make the inscriptions dark- used to treating poliomyelitis and measles colored, long-term and that the most famous among the Yoruba tribe of South Western solutions Serratia and Mahalbiah and the lack Nigeria. The seeds of henna have been of research or previous studies to assess reported to possess deodorant action and are health impact of these substances added to used in most cases of gynecological disorders henna. This study was conducted to such as menorrhagia, vaginal discharge and determine the effect of these additives to the leucorrhoea [1, 4]. The leaves of Lawsonia henna on the functions of the liver, kidneys, inermis with those of Hibiscus rosa-sinensis, and complete blood count in the white Eclipta prostrata and seeds of Abrus rabbits. precatorius when they are taken in equal Materials and Methods: quantities and ground into paste which is In this study, the using of the following soaked in sesame oil for 5 days is used as materials: hair oil by the tribes of Andra Pradesh, India Henna: (18). In Turkey, henna which is an extract of It was purchased from supermarkets in brack Lawsonia sp. is used as hair dye and nail dye city; known as (Hanna taj) which is a in many cultures as decorative dye centuries. powder, green in color Light placed in a Henna is widely used in the cosmetic paper bag weighs approximately 100 grams. industry as dyeing agent also in India [17].

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Mahalbiah and Serratia:  One rabbit was treated using 60 ml of They are unknown composition solutions Mahlbaih per 100 grams of henna. were added to the henna in order to increase Group 3 (Serratia): the darker color and worn on the body for a It included five rabbits average weight 1420 long time, was purchased from a ± 311.45 grams, was topical application of supermarkets in brack city. These solutions henna on the dorsal region of the rabbits after are oily liquid known as Mahalbiah a yellow hair removal procedure of which were color, Serratia with orange color, both placed covered treatment area using a transparent in glass vials each containing 20 ml of the nylon for 24 hours. Henna was mixed with solutions. water and added Serratia, which was divided Experimental animals: treatment of rabbits which are as follows: Only female were used during our present  Three rabbits were treated using 20 ml of investigation. The animals (n=15) were Serratia per 100 grams of henna. procured from stock animal facility of  One rabbit was treated using 40 ml of Department of Zoology, Sebha University, Serratia per 100 grams of henna. we divided it randomly into three groups  One rabbit was treated using 60 ml of (Control, Group 1, Group2) , five rabbits per Serratia per 100 grams of henna. group. The animals were acclimatized to the Methods: laboratory condition prior to treatment and The collection and separation of samples: given food and water. After topical application of Henna action for Group I (Control): 24 hours on the dorsal region removed them It included five rabbits average weight 1125 hair for rabbits have been slaughtered and ± 170.78 grams was topical application of assemble blood sampled and divided into henna on the dorsal region of the rabbits after whole blood collection tubes containing hair removal procedure of which were blocker clot EDTA in order to work full covered treatment area using a transparent picture of the blood, and the blood collection nylon for 24 hours and was used henna with tubes do not contain any anti coagulant to get water only . the blood, which was separated using a Group 2 (Mahalbaih): centrifuge speeds of 3000 r / min for 5 Included five rabbits average weight 820 ± minutes and then the separation of the serum 164.32 grams, it was topical application of in a dry and clean tubes to be used to henna on the dorsal region of the rabbits after measure AST , ALT, Urea and Creatinine. hair removal procedure of which were Statistical analysis: covered treatment area using a transparent All data was presented as means ±SD. nylon for 24 hours. Henna was used in this Statistical analysis was performed using T- group is henna mixed with water and added test. P-value < 0.05 was taken into Mahlbaih, which was divided by the consideration for determining significance. treatment of rabbits as follows: All statistical procedures were computed  Three rabbits were treated using 20 ml of using SPSS 10.0 software. Mahlbaih per 100 grams of henna. Results:  One rabbit was treated using 40 ml of As shown in Table (1) the mean of red blood Mahlbaih per 100 grams of henna. cells (RBC) in the first group (control) was 4.82 ± 0.125x1012 /L, in the second group

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(Mahalbiah) 5.35 ± 0.50 x1012 /L, in the third group (Control) was 16.5±11.7 U/L and the group (Serratia) 4.79 ± 0.211 x1012 /L .No second group (Mahalbiah) was 59.7±12.3 significant difference between control and U/L and the third group (Serratia) 64.0±14.2 Mahalbiah ,Serratia groups. Mean of white (table2). A significant difference was found blood cells (WBC) in the first group was between the first group and the second, third 10.70 ± 1.65x109 /L. in the second group 5.80 group (p-value ˂ 0.05). ± 1.85 x1012 /L; in the third group 7.37 ± The results of this study also showed that the 4.83 x109 /L. There was no significant average AST activity in the serum of rabbits difference between the first group and third of first group was 24.67±6.66 U/L, and the group (p-value ˃0.05), while the observed second group was 44.0±6.56 U/L, and the difference statistically significant among the third group was 75.0±13.7 U/L (table 2). A first group and second group (p-value˂ 0.05). significant difference was found between the The results showed also that the mean of first group and the second and third groups platelets (PLT) in the first group was 400 ± (p-value ˂0.05). 111 x109 /L, in the second group 337 ± 92.50 The results of this study showed that the x109 /L, in third group 339 ±271 x109 /L, average concentration of Creatinine in the there are no significant differences between serum of rabbits of first group (Control) was the first group and the second and third 0.80 ± 0.55 mg /dl and the second group groups. (Mahalbiah) was 2.16 ± 0.37 mg /dl and the Mean of hemoglobin (Hb) in the first group third group (Serratia) 2.50 ± 0.20 mg /dl was 10.93 ± 1.02 g/dl ,in the second (table2). A significant difference was found group11.67 ± 1.05 g/dl, in third group 10.60 between the first group and the second and ± 0.102 g/dl there are no significant the third group (p-value ˂ 0.05). differences between the control group and The results of this study also showed that the the two groups. The results showed that the average urea concentration in the serum of mean of size of red blood cells (MCV) in the rabbits of first group was 46.3 ± 11.2 mg /dl first group was 69.17 ± 3.86 ft, in the second and the second group was 79 ± 10.5 mg /dl group 65.43 ± 3.89 ft, in Third group 67.03 and the third group 87.7 ± 14.2 mg /dl (table ± 2.10ft . There was no differences 3). A significant difference was found significantly between the control group and between the first group and the second and second, third groups. third groups (p-value ˂0.05). The results of this study showed that the mean of ALT in the serum of rabbits of first

Table 1: Mean± SD of RBC, WBC, PLT, HG, MCV in three groups MCV(fl) HB (g/dl) PLT (x109 /L) WBC (x10 9 /L ) RBC(x1012 /L)

Control Mean±SD 69.17±3.86 10.93±1.02 400±111 10.70±1.65 4.82±0.125 Mean±SD 65.43±3.89 11.67±1.05 337±92.5 45.80±1.85 5.35±0.50 Mahalbiah p-value 0.3 0.4 0.5 0.04 0.2 Mean±SD 67.03±2.10 10.60±2.10 339±271 7.37±4.83 4.79±0.21 Serratia p-value 0.4 0.6 0.7 0.3 0.8

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elbaT 2: Mean ± SD of ALT, AST in three groups AST (U/L) ALT (U/L) Control Mean ±SD 24.67±6.66 16.50±11.70 Mean ±SD 44.0±6.56 59.70±12.30 Mahalbiah P- value 0.03 0.02 Serratia Mean ±SD 75.0±13.70 64.0±14.20 p-value 0.02 0.02

Table 3: Mean ± SD of Creatinine and Urea in three groups Creatinine (mg/dl) Urea ( mg/dl) Control Mean ±SD 0.80±0.55 46.30±11.20 Mean ±SD 2.16±0.37 79.0±10.50 Mahalbiah P- value 0.03 0.03 Mean ±SD 2.50±0.20 87.70±14.20 Serratia p-value 0.02 0.03

Discussion Group and Group added Serratia compared to Many chemicals are added to the henna to the group that used her only henna. increase the color dye from henna, Among It was expected to find a higher concentration the most famous substances that are added to of liver enzymes, Urea, Creatinin in both the henna in the Arab societies in general and group (Serratia and Mahalbiah) because it is the Libyan community in particular is not a natural material made with chemical Serratia and Mahalbaih ,a current properties [2, 18, 19]. commercial solutions in unknown markets But the results showed that there is also a rise source and unknown chemical composition . in the average concentration of liver Despite the widespread use of these materials enzymes, Urea, Creatinin in the results of significantly and frequently among women, rabbits that have been used with henna only but it has never conducted research or studies possibly due to the henna used henna on the impact of the use of such substances business may not be pure or contain on human health [10, 12]. additional materials undeclared to the This study was conducted to determine the consumer because it is supposed to henna impact of these materials on the functions of Sofa naturally is one of the medicinal plants the liver and kidneys and complete blood used in many treatments so we analyzed liver count of rabbits white with a dose of 20 ml of enzymes, Urea, Creatinin rabbits did not put mahalbiah and serratia per 100 gram of her henna at all were the results within the henna. normal range to them, and this can be In this study the henna is ready sold in confirmed in other research in the future known as the henna taj markets women using natural henna leaves are ground and employed on a permanent basis and processed in the lab [2, 3, 5]. frequently, the results showed that there is a When we changed the added dose of henna rise in the average concentration of liver in each of Serratia and Mahalbiah to 60 ml enzymes AST, ALT, as well as a rise in the per 100 gram of henna it led to animal death average of both urea and creatinine in both groups after about two hours of concentration in both the added Mahalbiah setting the mixture, it was noticed the deterioration of the case of animals once the

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Blend Mode them and put them in its own platelet (PLT) and (Hb) and the average size fund, where respiration rate began at least of red blood cells (MCV) with the exception gradually until she died about two hours later of a significant difference in (WBC) between and may have suffered a severe case of the first group (control) and seconed group poisoning. As well as the dose of 40 ml per (Mahalbiah). 100 gram of henna have also led to the death More research and studies to assess the of animals, but after a period of somewhat health impact of these substances that adding longer than the dose gram of henna 60 ml to the henna and promote health awareness per100 [3, 5, 11, 12, 19]. among women in Libyan society about this As for the results of CBC in the three groups harmful unknown chemical material is our there are no significant differences in the recommendations. average of red blood cells (RBC) and

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