Outcome of Fetoscopic Laser Coagulation in 61 Pregnancies with Twin-Twin Transfusion Syndrome in Viet Nam

Introduction

Given the increasing implementation of assisted reproductive technologies in the reproductive medicine, the incidence of twin pregnancies has been continuing to rise [1]. Thirty percent of twin gestations have monochorionic placentas which are susceptible to complications because of their unique placental architecture [2,3]. Twin-to-twin transfusion syndrome (TTTS) is one of the most severe complications of monochorionic twin pregnancies, accounting for 15% of twin pregnancies [3,4]. When transfusion syndrome occurs, the two fetuses share different portions of the same placenta and connect with each other through placental superficial vascular anastomoses [2], leading to the poor prognosis for both two pregnancies. Approximately 90% of twins acquired TTTS result in intrauterine demise of one or both fetus or perinatal death without any intervention [5]. About 50% of surviving twins are likely to leave irreversible neurological sequelae such as brain white matter necrosis, ventricular hemorrhage, cerebral dilation, cerebral palsy, neurocognitive impairment, etc [6-8]. The donor twin experiences the reduction of circulatory volume, anemia, decreased diuresis, oligohydramnios, hypotrophy. Meanwhile the recipient twin is characterized by circulatory hypervolemia, cardiomegaly, heart failure, fetal edema. Consequently, twins are at high risk of intrauterine demise or irreversible nerve damage after birth.
Treatment for TTTS includes methods such as amniocentesis, opening the amniotic septum between the two fetuses, fetoscopic laser coagulation (FLC) of the blood vessels connecting the two fetuses, or clamping the umbilical cord. Until now, the FLC and umbilical cord clamp are the only solutions to solve the pathogenesis of the TTTS. FLC was firstly conducted by De Lia at al. in 1990 under laparotomy and general anesthesia [9]. Subsequently, FLC has been improved and implemented in TTTS treatment worldwide [10]. It is demonstrated to be a beneficial therapy over serial aminoreduction in numerous population-based study as well as randomized controlled trial [4,10,11]. FLC has been also presented to reduce the long-term neurocognitive impairment in twins [12,13]. In Vietnam, Tam Anh Hanoi General Hospital is currently the only hospital that can perform FLC. However, there is no any study discussing the outcome of FLC in TTTS in Vietnam. Therefore, we conducted this study to evaluate the safety and therapeutic efficacy of FLC and other related factors in the outcomes of TTTS.

Methods

This is a prospective analysis study of pregnancies diagnosed with TTTS at the 16-26 week of gestation treated by FLC at Tam Anh Ha Noi General hospital, Vietnam. We included 61 consecutive pregnancies with moderate to severe mid trimester TTTS treated in our hospital of FLC between August 2018 and August 2019. This study protocol was approved by the Ethical Committee of Tam Anh Ha Noi General hospital on 1 August 1998. During the research period, all participants having TTTS confirmed by ultrasound and classified as the stage of II-IV (Quintero criteria) were extensively explained detailed risks and benefits of FLC. We only included participants accepting conducting the FLC and signing informed consent [14] [Box 1].

All procedures were performed under local anesthesia and ultrasound guidance. Firstly, the physician identified the position of fetoscope which is the most feasible position to approach the boundary line between the 2 amniotic chambers on the placenta surface from the amniotic fluid of the recipient fetus (Figure 1). In detail, this position has to allow the fetoscope parallel with the vertical axis of the donor fetus. Moreover, the attach positions of umbilical cords to placentas have to be observed clearly so that the conjunctive vessel will be detected more easily. Later then, the fetoscope (0°telescope, STORTZ) was introduced into the amniotic cavity of the recipient fetus and all the detected conjunctive vessels were coagulated along the whole vascular equator with a 0.4-0.6 mm fiber and laser system (20-40 watt of laser diot or laser YAGYttrium luminum Garnet).

The primary outcome was survival rate after 2 weeks of surgery and the live birth rate of one or both two fetuses. We also identified gestational age at delivery, the birth weight of neonate, the APGAR score and the weight of neonates seven day after birth as the secondary outcome variables. Gestational age in weeks was calculated based on the mother’s registration of her experienced due date. We divided gestation age at birth into very preterm (28- 31 weeks), moderate preterm (32-33 weeks), late preterm (34-36 weeks), early preterm (37-38 weeks), and full term (39-41 weeks). There was no post-term infants in the present prospective study. The birthplace at the Tam Anh hospital was only 21 pregnant women the remaining 40 pregnant women at the other 15 health facilities located in 14 provinces/cities nationwide.
Continuous variables are reported as mean and standard deviation (SD), categorical variables as absolute numbers and percentages. The differences in weight (percentage, %) was calculated by the following formulation ((weight of blood receiverweight of blood donor) *100/weight of blood receiver)) at the intervention time and the giving birth time, the end of gestation. Two-sided p<.05 were considered as significant using a t-test and odds ratio and 95% confidence interval. All statistical analyses were conducted with STATA Version 10.0.

Results

We recruited successfully 61 pregnant aged from 20 to 39, of which over 50% aged 25-29. Among them, 37 (60.7%) cases have reported having the first pregnancy, 17 (27.9%) cases having second pregnancy; 47 cases have no history of abortion and remaining 14 cases reported having abortion at least one time; 24 women have alive children and 37 other women with the first pregnancy. There was 52 women (85.2%) having history of caesarean section; 57 women (93.4%) have natural conception. 40.1% of case had anterior placenta, whereas 59.9% ones had posterior placenta. In included pregnancies, there were 26 cases having velamentous cord insertion. According to the Quintero staging classification, there was 19, 27, and 15 of pregnancies categorized to be stage II, III, and IV, respectively (Table 1).

Cervix length (mm) was 0-24 (eight cases), 25-29 (11 cases), 30-34 (21 cases), and 35-44 (21 cases). The difference (percentage) in weight (g) between two gestations was 1.33%-20% (12 cases), 20.1%-40% (32 cases), and 40.1%-62.23% (17 cases). The age at FLC (in week) was 16-20 (27 cases), 21-23 (24 cases), and 24-26 (10 cases). The number coagulated vascular anastomoses was 2-9 places in 34 cases and 10-23 places in 25 cases (Table 2). The estimated average (mean +/- standard deviation (SD)) of surgery time (minutes) was 35.3+/-11.1; of the volume (ml) of drained amniotic fluid was 1,210.7+/-817.6; of the percent (%) of differences between two fetal in weight was 30.9+/-13.6. At the intervention time, the estimated weight (g) of donor was significantly lower (280.5g+/-140.9g) than recipient (415.0g+/- 204.2g), (Appendix Table 1).

Among 61 women, 15 cases were miscarriage after the intervention within 14 days after performed FLC, remaining 46 cases being followed up. Nine cases reported having neonatal mortality before or after delivery. There were 37 cases having babies survived for 7 days, at the last study follow-up, Figure 2. Among 37 cases having successful live birth, Quintero II, III, and IV was 10 cases, 17, and 10, respectively. The proportion of participants who have no survivors was gradually decreased by increased Quintero staging, 47.4% (9/19), 37.0% (10/27), and 33.3% (5/15) for the Quintero II, III, and IV, respectively, non-statistically significant, p=0.394. The gestation age (in weeks) at delivery was full term of 39-41 (3 cases), earlier preterm of 37-38 (4 cases), late preterm of 34-36 (9 cases), moderate preterm of 32-33 (8) and severe preterm of 28-31 (13 cases), Table 3.

All 61 pregnant were healthy by the last follow-up time and 37 of them (60.7%) having at least one liveborn neonate, of which six cases gave the single birth of the donor fetus, eleven cases gave the single birth of the recipient fetus, and 20 cases had successful birth of both twins. Newborn survived for 7 days was 57 (46.7% of 122 gestation). There were not significant differences in birth weight between 6 single survived donors (mean 1,858.3g) and 11 single survived recipients (mean 1,954.5g), but significant differences in birth weight, among 20 survived twins, between donor twin (mean 1,410.0g) and recipient twin (mean 1,782.5g) in 20 cases of twins’ delivery, Table 4. The average weight (g) of the neonate was significantly lower in both the group of the gestation donors (1,918.8g versus 1,333.3g, p=0.012) and recipients (2,260.0g versus 1,763.5g, p=0.041) when compared the categorized Quintero II to the categorized Quintero III-IV, Table 5.

When compared the indicators of the first ten performed cases to the last-ten cases, from the first to the last 61 participants consecutively recruited, the average surgery time was longer (41.4 versus 33.0 minutes) but the average number of coagulated vascular anastomoses vascular anastomoses was lower (6.3 versus 10.7). Among 20 surviving twins, the estimated differences (in percentage) in weight between donor and receiver gestations have continuously getting worse and increased in two cases, from 25.09% and 6.83% (at the treated time) to 43.75% and 21.74% (at the end of gestation), respectively. This difference in weight between donors and recipients has decreased in the remaining 18 cases.

Discussion

The successful FLC in our study was over 60% after seven days of birth with severe TTTS. The performed intervention was the first time successfully treated in Viet Nam and the achievements and main findings are significantly contributing to the develop the treatment technique of FLC for further better managements of TTTS in Viet Nam and at low- and medium economy countries. The proportion of Quintero stage IV in our study population was two time higher than the previous four studies combined (24.6% (15/61) versus 12.3% (46/373)) [15-18], indicating big challenges to manage maternal and neonatal health in the present project. The higher proportion of the late stage of Quintero might be due to lack of established health facilities and trained physicians in the rural province in managing TTTS in the country. Therefore, the pregnant came to our hospital at the advanced stage of TTTS. Moreover, due to limited participants’ resources and poor quality of transportation from other regions in the South and Central areas to the Hanoi city, only one third (21/61) participants have completed the recommended monitoring and following-up at our hospital that might cause a poor outcome of the present pilot intervention project. Therefore, social-determinant factors of maternal and child health were possibly related to the present intervention study.
The average age at the intervention time (week) in the present study was similarly to the other five previous studies [15-18], from 20 to 22 week. The estimated average surgery time in the present study (35 minutes) was longer than the other recent studies in Germany and France (27-29 minutes) [18,19]. Due to local limited quality of disinfection, some pregnant women were suffering from infection after completed intervention (2/61) resulting the premature ended of the gestation. This unwanted outcome was not seen in the other studies [20]. The average gestation age of newborn of 37 cases in the present study was approximately 4 weeks lower when compared to the previous 953 cases of nine studies combined (29 versus 33 weeks) [15,16,20-26]. After the intervention, these pregnant often came back to follow their pregnancies at the local hospitals at disadvantage areas that might be related to the shorter period of gestation age. The deficiency of well-trained physicians in managing of TTTS could contribute to the high rate of preterm birth in our study. Furthermore, as in the low economic status, the hard labor, the malnutrition in these pregnant could be also the explanation for this. The overall survival of at least one surviving gestation in the present study (60.7%) was lower than previous four studies (69.2%-84.0%) [11,21,27-29].
Because there was not significant difference in weight (g) between single surviving donors and single surviving recipients but it was observed among 20 surviving twins, the possible explanation is that the nature of limited amount of blood due to unknown related factors in the donors or the velamentous cord insertion involvement. Fifteen participants have no survivor (all had miscarriage within 2 weeks of treatment) and most of them among the first half time of recruited participants due to our limited experiences. The other 9 cases had neonatal death might be due to poor taking care by other local health facilities outside of our hospital in Hanoi city. In our study, the mean estimated surgery time in the first 10 cases is shorter than the last 10 cases. After some first cases, our team gradually got more experience and spent shorter time to find the vascular anastomoses positions and do the coagulation. Treatment technique in TTTS would be transferred from the established health facilities of developed countries to the other in the low- and middle economy countries and this work would be coordinated by an international association and institution in taking care better maternal and child health. We assume that there is an estimated 3.4% of pregnancy is twin, of which about 33% is monochorionic; about 66% of monochorionic has the blood vessels connecting the two fetuses and about 15% of them developing TTTS [30], among about 140 million pregnancy in 2020 worldwide, there was about 155,509 TTTS globally.
In order to improve maternal and child health, global project to manage TTTS is highly needed. With about million newborn per year, Viet Nam will have about 1,111 pregnancy that will be suffering from TTTS and the findings of the present project is timely and that will contribute greatly to manage TTTS in the country. The present study certainly has limitations including a lack of followup for about two-third of participants due to social-economy difficulties, limited local health facilities. Another limitation is the lack of neurodevelopment follow-up of delivered babies. In spite of these limitations, during 13 months running of the project, we just managed for only about 5.5% of the estimated 1,111 pregnancy with severe TTTS and the work would be improved for further better management and outcome of TTTS in Viet Nam in particular and in other low- and median economy countries.

Acknowledgement

We deeply appreciate study participants and their family members with their cooperation in taking care maternal and child health to manage TTTS in the present study.

Conflict of Interest

There are no conflicts to disclose.

Box 1: Quintero’s staging.

Table 1: Characteristics of study population.

Table 2: Clinical Features when performed fetoscopic laser coagulation.

Appendix Table 1: Estimated indicators of twin-twin transfusion syndrome when treatment was performed.

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Oral PEY Treatment Induces Hair Regrowth and Improves Hair Quality in Patients with Alopecia

Introduction

Egg-yolk-based supplement have proven several biological beneficial effects and can be found in the market divided into unfertilized (commercialized) and fertilized. Scientific data demonstrated that eggs contain active compounds that may have a role in the therapy and prevention of chronic and infectious diseases. Although there is no currently scientific evidence to demonstrate it, eggs has been traditionally recommended for skin and hair problems and people have reported significant benefits after using eggs in their masks. However, the use of egg-yolk as a treatment of hair problems has stopped being used due to the appearance of more specific drugs and procedures to prevent hair loss and to improve hair regrowth. Among the most used procedures to combat hair loss nowadays, the transplant of hair with the FUE (follicular extraction unit) technique, is an innovative micrograft in which the hair follicles are transplanted together with all the perifollicular structures (including the blood vessels, the sebaceous gland or the piloerector muscle, among others) [1]. Furthermore, the major part of currently available conventional treatments are non-specific broad immunosuppressants administered locally or systemically, including topical and intralesional corticosteroids [2]. These treatments however are associated with multitude side effects such as skin atrophy, striae and telangiectasias, and even adrenal suppression, insomnia and glucose intolerance [3]. Other randomized controlled components under study include JAK inhibitors, such as tofacitinib, rollatini and baricite; fumaric acid esters; inhibitors of phosphodiesterases like apremilast; PGF2 analogs as Bigatures and Sterilant [4]. Nevertheless, in general, treatment of hair loss is often difficult and frustrating to patients and clinicians owing to the limited efficacy, the slow growth rate of hair and sometimes the adverse side effects.
In this study, we want to test the putative beneficial effects of our recently patented complement, PEY, in hair loss. PEY consists of an egg preparation obtained through a patented process from the fertilized egg-yolk [5]. A potent anti-inflammatory effect of PEY treatment has been recently reported in vivo [6]. Among the multitude of PEY components, highlight the presence of growth factors such as connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF-1) [6]; all of them with proven beneficial effects in the growth of hair follicles [7,8] Also, PEY exhibits a differential lipidomic and metabolomic profile with lipids clustered among different sphingolipid-related and immune-related pathways, and metabolites present in pathways related to omega-3 fatty acids, among others [6]. In addition to test PEY administration alone, we also want to analyze the putative efficacy of PEY to act as a booster with the most common-used treatments for alopecia.

Methods

All procedures and visits were conducted in accordance with the Guideline for Good Clinical Practice of the Institute Vila- Rovira. The study duration was around 4 months within which each volunteer (total of 45 patients) with alopecia was orally administered with 6 pills of PEY once a day. One group of patients (21 patients) only received PEY, whereas in the other group (24 patients) the PEY treatment was administered together with their usual treatment for allopecia. The PEY treatment consists of an egg preparation obtained through a patented process [5]. It comprises a mixture of yolk and white extracted from a fertilized egg that has been incubated for a short period, which then is lyophilized to obtain a commercial product called Excelvit. Qualitative analysis of the efficacy of PEY administration was determined by changes in global photography and with a satisfactory survey for each patient.

Results

Forty-Five Patients with a Clinical Diagnosis of Alopecia Were Enrolled in the Study

In the first group of patients (21 patients) PEY was administered alone to evaluate the efficacy of the product in alopecia. From this group, a total of 14 patients from the 18 that complete the treatment (77.7%) experimented a substantial improvement ranging from the stop in the hair loss to a better quality of the hair and skin. Notably, this effectiveness was higher in women than in men (86.41% vs 80%) (Table 1). In the second group of patients (24 patients) PEY was administered together with the usual alopecia’s treatment of the patient to analyze whether PEY can act as a booster to complement other treatments. In this case, from the 17 patients that complete the study 12 (70.58%) experimented an improvement of their condition ranging from hair regrowth Figure 1, stop of the hair loss Figures 2-4 and better quality of the hair Figure 3. Again, the efectiveness reported was higher in women than in men (76.92% vs 50%) (Table 1 and Figures 1,3,4). No side effects were detected by any of the patients in any treatment regimen along the study.

Table 1: Demographic and treatment variables of the study.

Figure 1: Patient demonstrated significant hair regrowth after the FUE transplant and PEY administration. This beneficial effect progressively continued up to 10 months of PEY treatment.

Figure 2: Patient after 3 months of treatment with PEY administered together with his conventional treatment for alopecia (PRP + Trichosol + Minoxidil 5%-F1% + Vit + Dutasteride). The patient experimented a reduction of hair loss, capillar regeneration and a faster hair growth. Also, the treatment provokes and increase in body weight.

Figure 3: Patient after 3 months of treatment with PEY administered together with her conventional treatment for alopecia (PRP + CAP Vitamin + Minoxidil 5%-F1%). The patient experimented a significant sttoping of the hair loss, capillar regeneration, a faster hair growth and a better hair texture.

Figure 4: Patient after 3 months of treatment with PEY administered together with her conventional treatment for alopecia (PRP + CAP Vitamin + Iraltone AGA plus). The patient experimented a complete sttoping of the hair loss.

Discussion and Conclusion

Our findings demonstrated that PEY treatment has a role in the treatment of alopecia. This treatment is effective when administered as a complement to conventional treatments for alopecia, but interestingly it has also a potent regrowing effect when administered alone. The anti-inflammatory effects already described for PEY, together with the higher CNTF and IGF-1 expression observed in cells treated with this compound, pointed to this molecule as potential candidates below the beneficial effects observed in patients (Cunill et al. Nutrients 2020). Indeed, both CNTF and IGF-1 has been linked to hair follicle growth and preservation [7,8]. Another highlighted point of this study is that the 73.3% of patients enrolled are woman, contrary to most of the studies of new drugs for alopecia that are focus exclusively or majority on men. Interestingly, the effects are proportionally higher in women than in men indicating that the hormonal control is one important aspect to take into account in the generation of new drugs. Remarkably, in contrast to major part of actual treatments for alopecia [2], no side effects were observed in any patient, probably due to the 100% natural origin of PEY.
In conclusion, this is an important study pointing towards the natural nutritional component PEY as a promising oral treatment for alopecia in both women and men and without any adverse side-effect. This study is in line with a growing data indicating that the food complements (like omega-3, curcumin, honey...) have a therapeutic effect through their anti-inflammatory and regenerative actions [9-13]. In this sense, it is essential that this compound will be introduced in the clinical practice, to increase the efficacy of human treatments, as will improve the general antiinflammatory state of the patients leading to a better resolution of medical interventions.

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Evaluation of an Active Butanol Fraction Effects of Blighia Unijugata (Sapindaceae) Leaves on Some Biochemical Blood Parameters in Male Wistar Rats

Introduction

Blighia unijugata (Sapindaceae), a species widespread in tropical and equatorial regions of Africa has been widely used for its multiple therapeutic properties [1]. In Côte d’Ivoire, Nigeria and Congo, this plant is abundantly consumed as vegetables and also used in the treatment of fever, nausea and vomiting, leprosy, eye pain, cough, migraines, rheumatism, kidney pain and joint stiffness, dizziness and especially high blood pressure [2,3]. However, despite the increased use of this medicinal plant, data-t-on its efficacy and safety are not yet available, thus exposing populations to all kinds of dangers. However, scientific studies and the rational evaluation of plants commonly used in traditional medicine could guarantee their best use, reduce the risk of accidents and allow the establishment of specific treatments for its poisonings [4]. Blighia unijugata has been the subject of several scientific publications due to its multiple uses in traditional medicine [5,6]. However, to our knowledge, very few scientific studies have been undertaken to establish the risks of toxicity of this plant on biochemical blood parameters. Previous work in our laboratory has shown that the butanolic fraction of Blighia unijugata (BFBu) has a significant hypotensive effect in rabbit of the species Oryctolagus cuniculus (Leporidae) compared to the other four fractions from the total ethanolic extract of this plant [7]. This study was therefore carried out to evaluate the effects of this active fraction on serum biochemical parameters of male Wistar rats.

Materials and Methods

Materials

Plant Material: The plant material consisted of powdered leaves of Blighia unijugata. The fresh leaves were collected in Abidjan (Cocody) in June 2009. This species was identified by us using available herbaria and a book written by [8]. The confirmation was made by the National Floristic Center (NFC) of Félix Houphouët-Boigny University (Abidjan, Ivory Coast) where a herbarium specimen has been kept under voucher no165.
Animals: The experiments were carried out on 24 albino rats (Ratus norvegicus), healthy male, of Wistar strain, aged three months. Their average body weight was 148.00±7.07 g. These animals came from the animal facility of the Institut Pasteur de Côte d’Ivoire located in Adiopodoumé (Abidjan) and were reproduced in the animal facility of the Laboratory of the Ecology Research Center of Nangui Abrogoua University (Côte d ‘Ivoire) at an ambient temperature of 25±3 °C. The photoperiod was 12 hours of light and 12 hours of darkness. Water and pellet food (Ivograin®) were fed ad libitum to the rats before the experiment began. These animals were treated according to good laboratory practices [9].
Technical Material : The extraction equipment for the total ethanolic extract and fractions of Blighia unijugata consisted of an electric grinder (Culatti, France), an electronic scale (Mettler Toledo, PB 153-L, Switzerland), a magnetic stirrer (Stuart SB 162, UK), Buchner funnel, cotton wool and Wattman no1 filter paper, Erlenmeyer flask, 500 mL conical flask, rotary evaporator (Büchi R110, Germany) and an oven (Retsch, Germany). The subacute toxicity study was performed using a gastric tube suitable for gavage of rats, pastor pipettes, and dry tubes. The analysis of the biochemical parameters was carried out using an automatic device (KENZA MAX Biochemis Try, France).
Solvents : The total ethanolic extract, hexanic, chloroform, ethyl acetate, butanolic and aqueous fractions of Blighia unijugata were obtained by extractions with different solvents (distilled water, 96% ethanol, hexane, chloroform, ethyl acetate and n-butanol).

Methods

Preparation of the Total Ethanolic Extract of Blighia Unijugata : The total ethanolic extract of Blighia unijugata leaves was prepared according to the method of some authors [10]. The fresh leaves were thoroughly washed with tap water and then dried at room temperature. They were sprayed using an electric grinder (Culatti, France). Cold maceration was carried out with 100 g of powder in 2 liters of 96% ethanol, for 48 h, with magnetic stirring. The resulting solution was first filtered through Buchner and then through Wattman No1 filter paper. This operation was repeated twice on the same powder residue. The filtrates obtained were added and concentrated under reduced pressure at 45 °C using a rotary evaporator (Büchi R110, Germany). The concentrated product, obtained after evaporation, was collected in a container and dried in an oven (Retsch, Germany) at 45 °C for 48 h according to the method described by [11]. The 13.3 g paste obtained corresponds to the total ethanolic extract (TEE) of Blighia unijugata. It was stored at -5 °C in a hermetically sealed jar.
Preparation of Fractions of the Total Ethanolic Extract of Blighia Unijugata : Different fractions of the total ethanolic extract (TEE) were obtained by successive liquid-liquid extractions, with four solvents of increasing polarities (hexan, chloroform, éthyl acétate and n-butanol) according to the methods of [12,13]. Ten grams of the TEE was dissolved in 200 mL of hot distilled water (45 °C). The whole was homogenized by magnetic stirring for 15 min and then it was filtered. Aqueous filtrate obtained was exhausted for 10 min at 27±2 °C with 200 mL of hexane, thus giving two phases after decantation (hexan phase and residual aqueous phase). Residual aqueous phase was again treated for 10 min at 27±2 °C with 200 mL of chloroform to in turn give two phases (chloroform phase and residual aqueous phase). Same operation was successively treating the residual aqueous phase with ethyl acetate to also give an ethyl acetate phase and residual aqueous phase, then with n-butanol to finally obtain butanol phase and residual aqueous phase after settling. Each organic phases and the residual aqueous phase obtained was recovered and concentrated under reduced pressure throught a rotary evaporator (Büchi R110, Germany). Various concentrated products obtained were collected in containers and dried in an oven (Retsch, Germany) at 45°C for 24 h according to the method of some authors [14]. Thus, hexan (0.6 g), chloroform (1.1 g), ethyl acetate (1.6 g), butanol (3.18 g) and aqueous (3.12 g) fractions were obtained. These end products were tested on rabbit blood pressure in the laboratory. Of these five fractions, butanol fraction coded BFBu had a greater hypotensive effect compared to the other four fractions. BFBu was therefore chosen for this study. The extractions were repeated several times in order to obtain a sufficient amount of extract to perform the tests. BFBu was stored at -5 °C in a tightly closed jar to prevent spoilage.

Methods of the Study of Subacute Toxicity

Preliminary Tests : The results of the acute oral toxicity study were used as the basis for the selection of the doses of BFBu that were administered to the rats. The initial dose level, selected on the basis of this orientation study, is well below the Maximum Tolerated Dose (MTD). At the end of these tests, the doses of 50; 500 and 1000 mg/kg bw were chosen to be the doses to use during this study.
Distribution and Treatment of Rats : Animals were divided into 4 homogeneous batches of 6 rats. This homogeneity of the different batches is a function of the body weight of the rats. The tests were carried out on a control batch and 3 batches of treated animals. According to the method described by [15], each rat from the control group received by gavage distilled water at 10 mL/ kg bw. Butanolic fraction (BFBu), diluted in distilled water was administered, by gavage, to each of the rats of the 3 test groups, respectively at doses of 50; 500 and 1000 mg/kg bw. Volume of FBu administered to the rats of each batch was less than 2 mL. Rats were given distilled water or BFBu daily by gavage every morning between 7 and 8 hours AM. GMT for 28 days.
Blood Sample : Days 7, 14, 21 and 28 of treatment, blood samples were taken on an empty stomach from the rats previously anesthetized with ether for 2 to 3 minutes, by puncture at the level of the orbital sinus of the eye, at the level of the eye using pastor pipettes according to the technique described by [16-18]. Thus, 2 to 4 mL of blood from each animal of the 4 batches were collected in dry tubes.
Determination of Biochemical Parameters : Automatic device (KENZA MAX Biochemis Try, France) was used for determination of the biochemical parameters. Blood collected in dry tubes was centrifuged at 3000 rpm for 10 min and the resulting serum was stored at -20 °C until assaying for biochemical blood parameters. Creatinine, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed by cinetic method [19]. Urea was determined by enzymatic method [19,20]. HDL (High Density Lipoprotein) Cholesterol Assay Method in VITROS HDL Plate is based on a non-HDL cholesterol precipitation technique followed by enzymatic detection [21]. Measurements of serum concentrations of total proteins, total cholesterol, triglycerides, bilirubins (direct and indirect) were carried out by the colorimetric method [19-23]. Glucose present in the serum was determined according to the glucose oxidase colorimetric method with the “Glucose Trinder GPO-POD” kit [20,24]. Finally, the electrolytes (magnesium, calcium, sodium, potassium and chlorine) were also determined by the colorimetric method [25-28].

Evaluation of the Atherogenicity Index

Atherogenicity index of rats was determined according to the formula of [29].
Atherogenicity index = total cholesterol / HDL − cholesterol

Statistical Analysis

Statistical analysis of data and graphical representations were performed using Graph Pad Prism 5.01 software (San Diego, USA). The results obtained were expressed as the mean followed by the standard error of the mean (M±ESM). Difference between the mean values of the biochemical parameters was determined by one-way analysis of variance (ANOVA 1) and supplemented by Tukey’s test for the comparison of the means of the parameters of the different test groups compared to the control group. The significance level was set at p<0.05.

Results

Effects of Bfbu on Biochemical Parameters

Effect of Bfbu on Creatinine Level : The mean creatininemie of the treated batch with BFBu at 50 mg/kg bw is lower than that of the control from D7 to D21 with the exception of D28 where it is higher than that of the control. D7, the creatinine level of the treated batch with BFBu at 500 mg/kg bw is lower than that of the control. However, from D14, this rate increases until the end of this study with a significantly (p<0.05) high rate with a value of 8.17±0.48 mg/L on D28. Only the creatinine level of the batch treated with BFBu at 1000 mg/kg bw increased throughout this study. This increase of 8.67±0.33 mg/L is significant (p<0.01) on the 28th day of treatment compared to the creatinine level of the control batch which is 6.17±0.60 mg/L (Figure 1A).

Effect of Bfbu on the Urea Rate: In the treated batches with BFBu at 50 and 500 mg/kg bw, the urea levels decreased compared to the control during this study, with the exception of D28 where there was an increase in this level. However, this decrease remains insignificant throughout this study. Moreover, from D7 to D14, in the treated batch with BFBu at 1000 mg/kg bw, a significant drop (p<0.05) in the urea level was noted compared to the control. Rate decreases and passes respectively from 0.21±0.02 g/L to 0.15±0.0 g/L and from 0.45±0.01 g/L to 0.37±0.02 g/L. However, from the 21st day of treatment, the amount of urea increased significantly (p<0.01-0.001) in this batch compared to the control batch. The urea rate increases and goes from 0.33±0.03 g/L to 0.54±0.01 g/L on D21 and from 0.35±0.04 g/L at 0.54±0.04 g/L on D28 (Figure 1B).
Effect of Bfbu on Total Cholesterol Level: Total cholesterol level of the treated batch with BFBu at 50 mg/kg body weight is lower than that of the control on D7 and D14. However, from D21, this rate undergoes a non-significant increase compared to the control. Moreover, the treated batches with BFBu at 500 and 1000 mg/kg bw, the total cholesterol level decreased during this study with the exception of the treated batch with BFBu at 500 mg/kg bw where this rate increased on D21. This decrease in mean cholesterolemia is significant (p<0.01-0.001) on D7 and D14 respectively compared to the mean value of the cholesterol level in the control batch, but it normalizes from D21. The cholesterol level decreases from 1.05±0.03 g/L to 0.85±0.03 g/L on D7 and from 0.99±0.03 g/L to 0.83±0, 03 g/L on D14 (Figure 1C).
Effect of Bfbu on Hdl-Cholesterol Levels: The mean HDL cholesterol values of the batch of rats treated with BFBu at 50 mg/ kg bw remained lower than that of the control during this study with a significant decrease (p<0.05) from a value of 0.27±0.01 g/L on D14. As for the HDL cholesterol level in the batch of rats treated with BFBu at 500 mg/kg bw, a decrease was noted during this work with the exception of D21 where its mean value is similar to that of the control. The mean value drops significantly (p<0.01) from 0.33±0.01 g/L to 0.27±0.01 g/L on D28 at this dose. Finally, in the batch treated with BFBu at 1000 mg/kg bw, the level of HDL cholesterol decreased throughout this study with a significant drop (p<0.001-0.01) in this value which went from 0.35±0.01 g/L at 0.30±0.00 g/L and from 0.33±0.01 g/L to 0.28±0.01 g/L respectively on D21 and D28 after administration of BFBu (Figure 1D).
Effect of BFBu on LDL-Cholesterol Levels: In the treated batch with BFBu at 50 mg/kg bw, the LDL-cholesterol level did not undergo any significant variation from D7 to D28 compared to that of the control batch In the treated batches with BFBu at 500 and 1000 mg/kg bw, the level of LDL-cholesterol decreased during this study. This drop is significant (p<0.05-0.01) on D7 and D14 in these two batches. From D21, BFBu does not cause any significant variation in the LDL cholesterol level with the exception of the treated batch with FBu at 500 mg/kg of bw where a significant drop (p<0.05) in this level was recorded on D28 with a value of 0.28±0.05 g/L (Figure 1E).
Evaluation of the Atherogenicity Index of BFBu: At a dose of 50 mg/kg bw, BFBu did not cause any significant increase in the atherogenicity index compared to that of the control group during this study. In the treated batch with BFBu at 500 mg/kg bw, there was a non-significant decrease on D7 followed by a significant decrease (p<0.05) in this index on D14 with a value of 2.77±0.16. From D21 to D28, the atherogenicity index of this batch remains similar to that of the control batch. Finally, in the treated batch with BFBu at 1000 mg/kg bw, the atherogenicity index remained low throughout this study compared to the mean value of the control batch. This drop is significant (p<0.05) on D14 where the mean value is 2.76±0.12 (Figure 1F).
Effect of BFBu on Triglyceride Levels: Repeated administration of BFBu at 50 mg/kg bw causes a non-significant decrease in the triglyceride level on D7 followed by an increase in this level from D14 until the end of this study. In the presence of BFBu at 500 mg/kg bw, the triglyceride level increases during this study with a significant increase (p<0.01-0.05) respectively on the 7th and 14th day of treatment. The respective average values recorded are 0.66±0.05 g/L and 0.50±0.01 g/L. In the treated batch with BFBu at 1000 mg/kg bw, a significant increase (p<0.01-0.001) in the level of triglycerides was noted respectively on D7 and D14 where the average levels are 0.64±0.01 g/L and 0.56±0.02 g/L. This increase is followed by a non-significant drop in triglyceridemia compared to the control batch on D21 and D28 (Figure 2A).
Effect of BFBu on Total Protein Level: Total protein level of the treated batch with BFBu at 50 mg/kg bw remained almost lower than that of the control during this study with the exception of D28 where this rate increased from 46.80±0.95 g/L at 48.70±1.05 g/L. Variations caused by BFBu at this dose are not significant (p≥0.05) compared to the control batch. In the treated batches with BFBu at 500 and 1000 mg/kg bw, an increase in this rate was noted throughout this work. This increase is significant (p<0.05) in the treated batch with BFBu at 500 mg/kg bw from D14. The increase in total protein level caused by BFBu at 1000 mg/kg bw is only significant (p<0.001) on the 14th day of treatment where its value is 61.50±0.43 g/L (Figure 2B).
Effect of BFBu on Alanine Aminotransferase Level: Alanine aminotransferase (ALT) level of the treated batches with BFBu at 50 and 500 mg/kg bw is lower than that of the control batch on D7. However, from D14 until the end of this study, the level of ALT increased in these batches compared to that of the control batch. The increase in this level in the treated batch with BFBu at 500 mg/kg bw is significant (p<0.001) from this period, whereas the increase in this rate is only exceptionally so on the 14th day in the lot treated with BFBu at 50 mg/kg bw where its value is 138±6.63 IU/L. In the treated batch with BFBu at 1000 mg/kg bw, the level of ALT, which remained almost the same to the control on D7, underwent a significant increase (p<0.001) from D14 to D28 with values of 165±2.63 IU/L (J14), 213±3.62 IU/L (J21) and 265±9.56 IU/L (J28) (Figure 2C).
Effect of BFBu on Aspartate Aminotransferase Level: On D7, all the doses of BFBu tested caused a significant decrease (p<0.05- 0.001) in the level of aspartate aminotransferase (AST) compared to the control. The mean AST value of the control batch is 188±7.92 IU/L. The AST levels recorded are 128±6.94 IU/L, 145±11.00 IU/L and 155±2.79 IU/L respectively for doses of BFBu of 50, 500 and 1000 mg/kg bw. However, from D14, an increase in the AST rate was recorded in all the batches treated with BFBu. This increase was not significant in all of the batch test from D14 to D21. On D28, in the treated batches with BFBu at 500 and 1000 mg/kg bw, a significant increase (p<0.01) in its mean value was recorded compared to that of the control batch (Figure 2D).
Effect of BFBu on Total Bilirubin Level: All tested doses of BFBu, the mean values of the total bilirubin level underwent a nonsignificant decrease (p≥0.05) compared to that of the control lot during this study with the exception of the lot treated with BFBu at 1000 mg/kg bw on D7 where this rate, a value of 9.50±0.34 mg/L, remained almost identical to that of the control (Figure 2E).
Effect of BFBu on Conjugated Bilirubin Level: With regard to the level of conjugated bilirubin, the mean values in the presence of all the doses of BFBu tested are lower than that of the control during this study. At 50 mg/kg bw, a significant drop (p<0.01-0.001) in the conjugated bilirubin level was recorded on D14 and D28, respectively, but this decrease was not significant on D7 and D21. At 500 mg/kg bw, the conjugated bilirubin level underwent a significant decrease (p<0.05-0.001) from the 14th day of treatment with the exception of D7 where this decrease is not significant with a value of 0.55±0.08 mg/L. Finally, at 1000 mg/kg bw, the level of conjugated bilirubin decreases significantly (p <0.05 - 0.001) during the treatment except on D21 when a non-significant drop in its value compared to the control was recorded. (Figure 2F).

Figure 1: Effect of Bfbu on Some Biochemical Blood Parameters and Atherogenicity Index.
Note: * p < 0.05; ** p < 0.01; *** p < 0.001; n = 6 : Differences were significant when values of treated groups with BFBu were compared to that of control group, at the same corresponding day.

Figure 2: Effect of Bfbu on Some Biochemical Blood Parameters.
Note: * p <0.05; ** p <0.01; *** p <0.001; n = 6: Differences were significant when values of treated groups with BFBu were compared to that of control group, at the same corresponding day.

Effect of BFBu on Glucose Level: No significant variation in blood glucose was recorded in the treated batches with BFBu at 50 and 500 mg/kg bw compared to the control during this study. On the 7th and 14th day of treatment, the mean values of the blood glucose level underwent a significant increase (p<0.05) in the treated batch with BFBu at 1000 mg/kg bw compared to the control batch. The average blood sugar values are 1.00±0.06 g/L (D7) and 0.85±0.02 g/L (D14). However, from the 21st to the 28th day of treatment, a significant decrease (p<0.05-0.001) in the blood glucose of the rats was observed in the treated batch with FBu at 1000 mg/kg bw compared to the mean value of the glycemia of the rats of the control group (Figure 3).

Figure 3: Effect of Bfbu on Glucose Levels.
Note: *p<0.05; ** p<0.01; ***p<0.001; n = 6: Differences were significant when values of treated groups with BFBu were compared to that of control group, at the same corresponding day.

Effects of BFBu on Electrolytes

Effect of BFBu on Sodium Level: Repeated administration of BFBu at doses of 50, 500 and 1000 mg/kg bw did not impacted with sodium levels during this study with the exception of the batch treated with BFBu at 1000 mg/kg bw where an increase significant (p<0.05) was recorded on the 28th day of treatment compared to the control batch. The sodium level is 143±1.31 meq/L at this dose (Figure 4A).
Effect of BFBu on Potassium Level: At 50 mg/kg bw, BFBu induces a drop in the potassium level with the exception of D28 where this rate increased compared to the control. These variations are not significant at this dose. At a dose of 500 mg/kg bw, the potassium level is lower than that of the control during this study. The drop in this rate is significant (p<0.001) on the 14th day of treatment in this batch where a value of 3.30±0.10 meq/L was recorded. However, on D21, the potassium level is higher than that of the control. As for the treated batch with BFBu at 1000 mg/kg bw, the potassium level remained lower than that of the control during this study with significant decreases (p<0.05-0.001) noted respectively on D7 and D14 but not significant of D21 to D28 compared to the control batch (Figure 4B).
Effect of BFBu on Chlorine Level: Chlorine level of the treated batch with BFBu at 50 mg/kg bw decreased significantly (p<0.05) on D14 compared to the control batch. As for the treated batches with BFBu at 500 and 1000 mg/kg bw, the chlorine level increased during this study with exceptionally significant increases (p<0.01-0.001) in this rate on D28 compared to the control batch. The respective values recorded at these doses are 122±3.53 meq/L and 119±1.50 meq/L. However, on D21, the chlorine level is almost identical in all the batches treated with BFBu (Figure 4C). Effect of BFBu on Calcium Levels: The calcium level of the treated batches with BFBu at 50, 500 and 1000 mg/kg bw did not undergo any significant variation (p≥0.05) during this study compared to the control (Figure 4D).
Effect of BFBu on the Magnesium Level: On D7, the magnesium level of the treated batches with BFBu at 50 and 500 mg/kg bw is higher than that of the control. However, from the 14th day of treatment, this rate decreased in these batches compared to the control. Only the magnesium level of the treated batch with FBu at 1000 mg/kg bw continuously decreased during this work. All the variations caused by BFBu at the doses tested remained insignificant (p≥0.05) compared to the control during this study (Figure 4E).

Figure 4: Effect of Bfbu on Electrolytes.
Note: p> 0.05; n = 6: non-significant difference compared to the control batch
* p <0.05; ** p <0.01; *** p <0.001; n = 6: Differences were significant when values of treated groups with BFBu were compared to that of control group, at the same corresponding day.

Discussion

The various biochemical parameters examined in this study are useful indicators for assessing the toxicity of plant extracts in animals [30]. Analysis of blood parameters is relevant in risk assessment since changes in the hematologic system have a very high predictive value for toxicity in humans [31]. The significant reduction in the level of transaminases (AST and ALT) observed at the start of the experiments indicates that the absorption of BFBu does not cause cytolysis but rather a protective effect in the liver. BFBu does not harm the liver within the first week of administration. However, at the end of this study, the increase in these parameters following the administration of BFBu could be due to the appearance of necrosis in one or more organs, namely the liver, heart and kidneys, thus causing an increase in the level of these enzymes in the blood. Indeed, according to [32], damaged tissue is generally associated with the release of specific enzymes from the tissue, or the affected organ, into the bloodstream. The consequence is an increase in the activity of such enzymes in bodily secretions. ALT is a cytoplasmic enzyme found in very high concentrations in the liver, and an increase in this specific enzyme suggests liver cell damage while AST is less specific than ALT as an indicator of liver function. The increase in the level of transaminases (ALT and AST) has also been observed in liver disorder [33-36]. These results are in agreement with those of [37,38] who reported that the ethanolic and methanolic extracts of Alchornia cordifolia leaves cause an increase in the activity of transaminases. These authors therefore suggested that these extracts exhibit hepatotoxic effects at doses of 250 to 500 mg/kg bw and 800 to 1600 mg/kg bw, respectively. BFBu would therefore be, at the tested doses of 500 and 1000 mg/ kg bw and in the long term, probably poorly tolerated by the liver. According to [39], the increase in the serum total protein level is an indication of tissue damage while the significant decrease in the total protein content of the liver is a reflection of hepatic nontoxicity. The significant increase in the total protein content in the batches of rats treated with BFBu at 500 and 1000 mg/kg bw could therefore indicate an increase in protein stores and therefore suggest hepatic toxicity, which would again confirm the detrimental effects of BFBu at 500 and 1000 mg/kg bw. Bilirubin, a metabolic breakdown product of heme derived from senescent red blood cells, is also one of the most commonly used tests for liver function. Its concentration could indicate the condition of the liver and the type of liver damage [40,41]. The significant reduction in total and conjugated bilirubin levels could be provided by a deficiency in the secretory function of these proteins. Certain substances present in FBu could therefore imply inhibitions at the level of the hepatic cells. This reduction can also affect the functional activity of the liver. Doses of BFBu of 50 and 500 mg/kg bw did not significantly affect blood sugar. Only the dose of 1000 mg/kg bw increased it on D7 and D14 and decreased on D21 and D28. These results suggest that this fraction has hyperglycaemic activity followed by hypoglycaemic activity at high doses. At the end of this study, the hypoglycaemic activity could be attributed to the existence of the molecules demonstrated in this fraction. Indeed, flavonoids from different plants have shown a promising hypoglycemic effect in diabetic animal models [42-44]. Saponins are triterpene, steroidal or alkaloidic glycosides. Authors have shown a hypoglycemic activity of triterpene glycosides [45,46]. It is therefore possible that the presence of flavonoids and saponins in FBu could be responsible for the hypoglycemic effects of this fraction. Some authors reported a significant decrease (p<0.05) in the fasting blood glucose level of rats made diabetic with alloxane when given the ethanolic extract of Ficus microcarpa, orally for two weeks [47]. Finally, these results are in agreement with the studies conducted respectively on the seed cotyledon and leaves of Chrysophyllum albidum at doses between 250 and 1000 mg/kg bw [48,49] on the one hand, and on the other hand on Bixin (10 mg/kg bw) extracted from the seeds of Bixa orellana [50]. These authors have shown that these extracts significantly decrease the glucose level in normal and diabetic rats induced by alloxane. However, the notable increase in blood sugar observed in this study could be linked mainly to the stress undergone by the animals at the time of sampling as suggested by [51] during work with Momordica charantia in rabbits. However, a real hypoglycemic effect could be attributed to this fraction by carrying out further study on suitable models. The significant (p<0.01) increase in mean serum creatinine was seen in the lots that received the highest doses of FBu (500 and 1000 mg/kg bw). This result suggests that BFBu is toxic to the kidneys. Creatinine is the major catabolic product of muscles and is secreted by the kidneys. Serum creatinine levels are used as an indicator of kidney defects [38,52,53]. The significant drop in the urea level observed at the start of treatment in the batch treated with FBu at 1000 mg/kg bw would be a sign of the good functioning of the kidney and the lack of renal toxicity due to this fraction. However, the increase in this level in this batch would be an indication of azotemia. According to Nduka [54], the high urea level is associated with the increased catabolism of tissue proteins. The excess protein in the blood collapses and urea excretion decreases, increasing its level in the blood. The increase in this level would also be a sign of damage to the kidney, which can no longer extract nitrogenous excretion products from the blood and concentrate them in the urine, thus confirming the renal toxicity caused by BFBu. Other indicators of kidney function such as sodium, potassium and chlorine were significantly affected by BFBu except calcium and magnesium which were not. The urea, sodium and chlorine levels significantly increased in the treated batches with BFBu at the highest doses during this study. These results probably suggest nephrotoxicity of BFBu at high doses, which would support the hypothesis made about the increased creatinine level. However, the lack of a significant effect of BFBu on serum calcium and magnesium concentrations in animals suggests that the secretory capacity of the kidney and normal organ function related to these parameters were not affected. According to the work of [41] on the aqueous extract of Felicia muricata at doses of 50, 100 and 200 mg/kg bw, the calcium and magnesium levels of the rats were not significantly modified. The hypokalaemia caused by repeated administration of BFBu may be due to an alteration in tubular reabsorption of potassium. Similar effects have been reported by [55] with the aqueous extract of Arctotis arctotoides on the decrease in potassium concentration. These authors suggested that this plant would not cause any damage to the cardiovascular system, which could also be the case with BFBu. Changes in the concentration of major lipids such as total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides, as well as the atherogenicity index may provide useful information on lipid metabolism and heart predisposition, atherosclerosis and its associated coronary heart disease [29,56-58]. In this study, the decrease in serum cholesterol level could be explained by the deterioration of cholesterol biosynthesis. This has also been suggested by [59] who showed that the aqueous extract of Chrysocoma ciliata at doses of 50, 100 and 200 mg/kg body weight decreases the cholesterol level in rats. Elevated cholesterol concentration is an important risk factor for cardiovascular disease [60]. Therefore, lowering cholesterol at the doses of BFBu tested could be clinically beneficial as this fraction is unlikely to be associated with cardiovascular risk. Triglycerides are the storage forms of fatty acids. The decrease in the level of lipids such as HDL and LDL cholesterols as well as the decrease in the atherogenicity index may be an indication that FBu would not predispose animals to the risk of atherosclerosis and associated coronary heart disease. According to several authors, the total cholesterol/cholesterol- HDL ratio constitutes a revealing index of arterial and especially coronary risk. This ratio would indicate an increased risk if it is greater than 4.4 [29]. However, according to the results obtained during this study, the atherogenicity index of each animal was lower than this value. BFBu could therefore be protective of heart tissue. Furthermore, the decrease in serum lipid parameters investigated in this study suggests that the lipid metabolism of animals was disturbed by the administration of BFBu. This disturbance could be an indication that BFBu would probably not predispose the animal to atherosclerosis and associated coronary heart disease despite the notable elevation (p<0.001) in triglyceride levels during the first 14 days in batches treated with BFBu at 500 and 1000 mg/ kg bw.

Conclusion

This study showed that butanol fraction of Blighia unijugata (BFBu) produced changes in biochemical parameters following repeated administration. BFBu caused disruption of the carbohydrate, lipid and protein balances. At high doses, BFBu caused hepatic and renal toxicity. However, the fraction tested at dose of 50 mg/kg bw, is non-toxic. This fraction has hypoglycemic effects and could be used in the management of diabetes, respectively. BFBu would not predispose rats to the risk of atherosclerosis and associated coronary heart disease.

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