Bixin and Annatto seeds Extract maintains ADM levels to Sustain Pregnancy
K. R. Padma, P. Josthna*
Department of Biotechnology, Assistant Professor, Sri Padmavati Mahila Visva Vidyalayam
(Women’s University), Tirupati, Andhra Pradesh, India.
*Corresponding Author E-mail: thulasipadi@gmail.com
ABSTRACT:
Bixa orellana (family Bixaceae) is a neotropical rapid growing perennial tree of immense agro-industrial value since its seeds have a high carotenoid content, chiefly bixin. It has been utilized since pre-colonial times as a culinary colorant and spice, and for healing purposes. It is at present used as a natural pigment in the food, in pharmaceutical and cosmetic industries, and it is commercially acknowledged as annatto. We have investigated the antioxidant property of bixin which have the capability to reduce blood pressure during pregnancy and maintain ADM levels to sustain pregnancy. A diet rich in bixin and iron content sustain pregnancy in rats. The present study aimed to evaluate the effect of the treatment with aqueous extract of annatto seed and isolated bixin on the rats. Similarly to evaluate the importance of adrenomedullin hormones, we have injected rats with ADM22-52 antagonist. Experimental group of Rats especially Group-I and II on the gestational day 2 were implanted subcutaneously with osmotic (Alzet) minipumps delivering 125 and 250 μg rat/day/of AM22–52 and were killed on the gestational day 9. Group-III experimental rats was administered orally with bixin during early pregnancy and group-IV was control. Our results demonstrated that rats administered orally with isolated bixin compound, sustained pregnancy and ADM antagonist injected group showed fetal resorption. We have ascertained the hypothesis that ADM, a multiregulatory ubiquitous peptide hormone, works as a trophoblast pro-invasion factor, as a growing body of evidence which clearly signifies that bixin enhances ADM levels which in turn helps to sustain healthy pregnancy to full term baby.
KEYWORDS: Adrenomedullin, Bixin, annatto seed, carotenoid, pregnancy, ADM antagonist, fetal resorption.
INTRODUCTION:
Aboriginal people still use the pulp for 'cosmetic purposes', as hair dye or lip stick, for this reason the English common name 'Lipstick tree' originated. The pulp is also said to repel insects and to protect against sunburn due to the UV-filtering properties of the carotenoid pigment known as Bixin (5). The ancient history reveals the whole tree as a valued medicinal plant that has been used to treat a wide variety of conditions from fevers to cancer (Bixa orellana).
Moreover to its traditional exploits in tropical America, annatto has also been in use worldwide as a color additive for foods, drugs and cosmetics. As a food color additive, annatto has been expansively used for coloring high-fat dairy products such as butter and cheese, and for a mixture of other products such as cereals, margarines, sausages, snack foods, coffee creamers, ice-cream and seasonings. Bixin, a carotenoid devoid of pro-vitamin A activity, is the main oil soluble pigment found in annatto (6). Bixin rich diet is crucial during pregnancy.
The carotenoids on the whole commonly found in plant foods are the B-carotene (carrots, Daucus carota), lycopene (tomatoes; Lycopersicum esculentum), numerous xanthophylls (zeaxanthin, lutein and other oxygen corn structures, Zea mays, of mango, Mango indicates; of papaya, Carica papaya and egg yolk) and the bixin (annatto, Bixa orellana) (7). Annatto is one of the mainly used natural dyes, and Brazil is one of the leading producers and exporters of annatto seeds. Formerly, ADM has been concerned in a multiplicity of reproductive functions, specifically during pregnancy and also has concerned interest in reduction of feto-maternal exchange of oxygen by using ADM antagonists (8-11).
MATERIALS AND METHODS:
Animal maintenance:
Animal studies were performed as per institute animal ethics committee regulations and approved by the committee (Reg. No. 1677/PO/a/12/CPCSEA/SPMVV-IEC/2014/01). Female Wistar rats with 220-250 g weights were used for this study. Rats were placed in plastic cages covered with wire mesh. Temperature was maintained at 37oC and a humidity of 55±5 % under a 12h light-dark cycle. Healthy female rats of Wistar strain were purchased from authorized vendor (M/S Raghavendra Enterprises, Bangalore, India).
Experimental Treatment Protocol:
Female Wister rats (Rattus norvergicus), weighing 220g ± 280 g were used for the experiment. Three to four pregnant rats were used for each of the experimental study. Female rats were mated with male proven breeders. The next day morning, collecting of vaginal secretion with a plastic pipette filled with 10 μL of normal saline (NaCl 0.9%) by inserting the tip into the rat vagina. One drop of vaginal fluid was placed on glass slides the unstained material was observed under a light microscope (16-17). Two females of pro-estrous stage were paired with a male overnight and the next morning, males were removed and females were assessed for the presence of sperms in the vaginal flush. Animals with positive sperm in the flushes are designated as day 1 of gestation.
Fig-1 Collection of Vaginal Secretion
Treatment:
The seeds of the plant were dried and powdered. The Annatto powder suspended in corn oil (100 mg/ml) was administered by gavage to rats once a day from days 2–8 of pregnancy. A control group received only saline. All rats were weighed on days 9 of pregnancy and sacrificed.
Carotenoids:
The main compounds found in B. orellana plant are carotenoids and apocarotenoids. Numerous phytochemical studies have been carried out on isolation and identification of carotenoids and apocarotenoids of several extracts. Majority of the carotenoids have been isolated from seed and seed coats. Bixin (18) [methylhydrogen-(90 Z)-6,60 -diapocarotene-6,60 -dioate] is the chief carotenoid compound present in B. orellana seed coat and reports for 80% in addition to the presence of other carotenoids in trace amounts (19,20). (Shown in fig-2 and 3).
Fig. 2. (a) Plant (b) leaves and flower, and (c) seeds and dye.
Figure-3: Annatto seeds
Osmotic (ALZET) pumps:
The mean ± SEM body weight was 228 ± 260 g in the rats on day 2 of gestation. The osmotic minipumps (model 2001 Alzet pump; 1.0 μl/h were inserted subcutaneously into the dorsum of pregnant rats while the animals were under anaesthesia (anaesthesia consisted of a combination of ketamine (45mg/kg) and xylazine (5 mg/kg). The minipumps were filled with saline alone or with saline containing different concentrations of AM22–52. These concentrations were chosen based on the earlier findings of Witlin et al.,(21)and Penchalaneni et al., (22,23) to deliver AM22–52 at 125 and 250μg/rat/day.
Statistical analysis:
Uterus and implantation weights were performed by taking the mean of the uterus and implantation weights per rat first followed by the mean of all the rats. Weights are expressed as the mean ± SEM and were analyzed for differences using one way ANOVA. Values were found to be significant when *P< 0.05.
Results
Dose dependent effects of Adrenomedullin antagonist and Bixin on uteroimplantation growth weights:
This portion of the study evaluated the uteroimplantation growth effects of AM antagonist infusion initiated on gestational day 2 and continued through gestational day 8 as compared with vehicle control. The oral administration group with bixin compound did not show any effect on body weights. The detrimental effects of AM antagonist infusion appeared to manifest in both the uterus and implantation which is determined by significantly decreased uterus and implantation growth during the dose response effect experiment as shown in table-1.
Table-1 Uterus and implantation sites weight for rats killed on gestational day 9
Treatment group |
pregnant rats per group (n) |
uterus weight (g) |
implantation wt (mg) |
Control |
04 |
42.6±0.9 |
8.92±0.1 |
Bixin (100mg/ml) |
04 |
42.6±0.9 |
8.92±0.1 |
125µgAM22-52 |
04 |
39.4±0.3 |
7.95±0.1 |
250µgAM22-52 |
04 |
22.1±0.7 |
5.55±0.3 |
Fig-4 Uterus and Implant weight of treated rats
Adrenomedullin in fertility and implantation:
An important role of AM in fertility and implantation has come from well-characterized animal models. Recent findings have implicated AM in even the earliest stages of pregnancy. Padma et. al., (2016) showed that in a rat model, ovarian AM expression increases from small antral follicles to large antral follicles to the formation of the corpus luteum, and AM appears to be involved in the regulation of progesterone production from the corpus luteum (24). In the present work carried by us we observed in (figure-5) foetal resorption sites, when physiologic and environmental factors varied which resulted in resorption sites in early pregnancy.
(a) (b) (c)
Fig-5 Control and treated groups (a) Control (b) 125 µg AM22-52 and (c) 250 µgAM22-52
DISCUSSION:
The development of an annatto extract, with a rather high content of bixin, was administered in rats orally with doses up to 100 mg/ml/day on days 2–8 of pregnancy. Since no sign of toxicity had been noted in a preliminary dose-range-finding experiment, No adverse effect of annatto on the mother was observed. Annatto did not induce any increase in embryolethality, neither did it cause any embryofetal growth retardation. ADM is responsible for healthy pregnancy. Therefore a diet rich in bixin enhances the levels of adrenomedullin. Any aberration due to environment detoriation may detract in implantation. Furthermore, additional in vitro and in vivo methods are employed to gain a greater understanding of the mechanisms by which AM mediates its effects in reproductive biology, with the intention of linking the biological functions of the peptide to physiologically relevant paradigms such as host defense, vasodilation, angiogenesis or regulation of innate immunity.
We have used the time-course and dose-response studies to evaluate the complete spectrum of ADM antagonist effects. In every variation of ADM antagonist exposure, deleterious effects to the uterioimplantation regions were observed. The uterus and implantation weights showed apparent decrease in 250 μg/rat/day than in control, bixin and low doses. Adrenomedullin is a multiregulator that put forth its effects through dual pathways in each of two vascular cell types.
CONCLUSION:
Several mechanisms have been illustrated in the pathophysiology of pregnancy complications, such as intrauterine growth retardation, gestational hypertension, and preeclampsia (PE). We entail that one such mechanism may be increased apoptosis caused by decrease in the levels of ADM during pregnancy. There are a massive amount of genetic, physiologic, and environmental factors that must all work together in perfect harmony throughout pregnancy to generate the magic so-called “miracle” that is a healthy well-growth baby. Any aberration in this process may result in pregnancy complications, which can include implantation failure, miscarriage, fetal growth restriction, gestational diabetes, PE, and preterm birth. Given in this complexity, the bixin compound has the ability to sustain pregnancy and this created a interest to provide a diet rich in bixin during pregnancy to increase ADM levels and prevent resorption. There is a currently major interest in ADM and efforts have been set forth in the field, to expand our understanding of the factors that contribute to healthy versus unhealthy pregnancies.
Ethics Approval and Consent to Participate:
Animal studies were performed as per institute animal ethics committee regulations and approved by the committee (Reg. No. 1677/PO/a/12/CPCSEA/ SPMVV-IEC/2014/01).
ACKNOWLEDGEMENTS:
The authors express their appreciation to Sri Padmavathi Mahila Visvavidyalayam (Women’s) University for providing access to the research facilities and for actively participating in the study and also thanks to the faculty, staff, and students from the Mahila University for their assistance in the research studies.
COMPETING INTERESTS:
Not applicable.
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Received on 21.11.2018 Modified on 12.12.2018
Accepted on 06.01.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(3): 1315-1319.
DOI: 10.5958/0974-360X.2019.00220.8