Maternal obesity, environmental factors, cesarean delivery and breastfeeding as determinants of overweight and obesity in children: results from a cohort

Daniel S Portela
Tatiana O Vieira
Sheila Ma Matos
Nelson F De Oliveira
Graciete O Vieira
Maternal obesity, environmental factors, cesarean delivery and breastfeeding as determinants of overweight and obesity in children: results from a cohort
10.1186/s12884-015-0518-zR E S E A R C H A R T I C L E Open Access
Background: Overweight and obesity are a public health problem with a multifactorial aetiology. The objective of this study was to evaluate risk factors for overweight and obesity in children at 6 years of age, including type of delivery and breastfeeding.Methods: This study relates to a cohort of 672 mother-baby pairs who have been followed from birth up to 6 years of age. The sample included mothers and infants seen at all ten maternity units in a large Brazilian city. Genetic, socioeconomic, demographic variables and postnatal characteristics were analyzed. The outcome analyzed was overweight and/or obesity defined as a body mass index greater than or equal to +1 z-score. The sample was stratified by breastfeeding duration, and a descriptive analysis was performed using a hierarchical logistic regression. P-values of <0.05 were considered significant.Results: Prevalence rates (PR) of overweight and obesity among the children were 15.6% and 12.9%, respectively. Among the subset of breastfed children, factors associated with the outcome were maternal overweight and/or obesity (PR 1.92; 95% confidence interval "95% CI" 1.15-3.24) and lower income (PR 0.50; 95% CI 0.29-0.85). Among children who had not been breastfed or had been breastfed for shorter periods (less than 12 months), predictors were mothers with lower levels of education (PR 0.39; 95% CI 0.19-0.78), working mothers (PR 1.83; 95% CI 1.05-3.21), caesarean delivery (PR 1.98; 95% CI 1.14 -3.50) and maternal obesity (PR 3.05; 95% CI 1.81 -5.25).Conclusions: Maternal obesity and caesarean delivery were strongly associated with childhood overweight and/or obesity. Lower family income and lower levels of education were identified as protective factors. Breastfeeding duration appeared to modify the association between overweight/obesity and the other predictors studied.

Background

Overweight and obesity are a public health problem in both developed countries and those in development [1]. Prevalence rates of excess weight in Brazil have been increasing over recent decades [2].

Studies performed in Brazil at the end of the last century have shown a prevalence varying from 10.8% to 26.2% for childhood overweight, and 7.3% to 8.5% for childhood obesity [3,4]. A decade later, research has shown that the prevalence of childhood overweight varies from 15.3% to 34% [5,6].

The multifactorial aetiology of obesity is consensus in the literature, involving genetic, environmental, socioeconomic and behavioral factors [4,7]. In view of the difficulty of treating the condition, identification of risk factors, and most importantly those that can be modified, provides epidemiological tools of fundamental importance for planning intervention measures with a positive impact on public health.

The literature discusses the association between factors present in the fetal period, neonatal period, and first months of life with childhood and adult diseases [8][9][10][11][12]. Many studies suggest that breastfeeding may be a protective factor against childhood obesity [6,11,[13][14][15][16]. The lower protein content of breast milk is related to a lower risk of obesity in childhood. Furthermore, the practice of breastfeeding has a protective effect through the protein quality, for example the alpha-lactoalbumin [17], the auto regulation of volume ingested, and the improved acceptance of healthier foods [13,18]. However, some researchers question the protection of breast milk against childhood obesity [19].

Similarly, other determinants of risk in earlier phases of life are currently being studied, for example, caesarean deliveries. However, studies investigating this association are scarce [8,20].

The objective of this study was to evaluate the association between environmental and genetic risk factors, type of birth, and overweight and/or obesity at 6 years of age.

Methods

Study design

This was a cohort study conducted in the city of Feira de Santana, which is 108 km from the state capital of Bahia, in Northeast Brazil.

Population and study sample

The children enrolled on this study are part of a cohort that began in April 2004, comprising of all live births at all ten maternity units in the municipal district.The following exclusion criteria were used in this study: mothers who were separated from their children by the justice system (for example, imprisoned); children with diseases that contraindicate breastfeeding; those who live in locations that present a risk to the safety of the interviewer (for example, due to drug trade, prostitution).The sample size calculation for this study was based on a 6.0% estimated prevalence of overweight among children under 5 years old in the Northeast of Brazil [21], a level of precision of around 1.25% and 95% confidence, which resulted in an estimated sample of 526 children. This figure was increased by 30% to cover predicted losses, resulting in a final sample size calculation of 684 children. However, data from 672 children followed in the cohort from birth to 72 months were actually analyzed.

Data collection

Questionnaires were administered by trained health professionals. The data analyzed were collected during the first 72 hours after delivery, during monthly home visits for the first six months of the children's lives and during interviews conducted 9, 12, 36 and 72 months after the birth of each child.

Variables

Body weights of mothers and their children were measured in triplicate at the 72-month home visit, using a digital balance with precision of 100 gram and a maximum capacity of 150 kg. Height was measured using a portable and dismountable stadiometer with a built-in platform and a maximum height of 216 cm.

Anthropometric status was assessed using body mass index (BMI) at six years of age, calculated using World Health Organization's (WHO) Anthro Plus software, which uses the WHO growth curves as a reference [22,23]. Children were classified by nutritional status as underweight or normal weight if BMI was below a z-score of +1 or as overweight or obese if BMI was greater than or equal to a zscore of +1.

The variable of mother's educational level was dichotomized with a cut off of 8 years, equivalent to middle school in Brazil, as this is the cut off commonly used by other Brazilian studies. The variable of mother's age was dichotomized between adolescence (<20 years) and adult. Family income and sedentary activities were dichotomized according to criteria of convenience, based on measures of central tendency and dispersion.

The remaining variables analyzed were subdivided into three different groups, as follows, 1. socioeconomic and demographic characteristics: mother's educational level, mother's age, family income, mother was going out work at time of birth; 2. genetic characteristics: maternal BMI; 3. children's neonatal characteristics: type of delivery, sex, birth weight, gestational age, and postnatal factors, as their lifestyle habits at six years of age, such as physical activity at school; total time spent daily in sedentary activities such as watching television, playing videogames or using a computer; sports outside of school; playing ball games, riding a bicycle and buying school meals.

Statistical analysis

Initially, sample characteristics were compared by breastfeeding in its original continuous scale using Student's t test. Effect modifying factors were then evaluated and stratified by breastfeeding, and the Pearson's chi-square test (x 2 ) was used to analyze factors associated with overweight/obesity. For all tests, a p-value of <0.05 was considered significant.

Logistic regression analysis was conducted on the two strata of children classified by breastfeeding duration, with insertion of variables according to a three-level hierarchical theoretical model (Figure 1), and variables distributed according to their proximity to the outcome: socioeconomic and demographic characteristics (model 1, distal level), genetic characteristics (model 2, intermediate level) and characteristics of the neonatal and postnatal period (model 3, proximal level).

All variables that attained a p-value of ≤ 0.10 in bivariate analysis were included in the hierarchical analysis. Variables that attained an intralevel statistical significance of 5% were then used to adjust the following level, and this procedure was repeated successively until inclusion of the last eligible variable in level 3, with definition of factors associated with overweight/obesity among children under six years to a significance level of 5%. The model's fit was then tested. Analyses were conducted using SPSS 10.0 and R statistical software packages [24]. The same procedure was then conducted for two strata of children separated by breastfeeding duration.

Ethical aspects

The study has been submitted and approved by the Ethics and Research Committee of the State University of Feira de Santana, Bahia, Brazil under Protocols 12/2003 and 077/2006. All women included in the study provided informed written consent.

Results

General characteristics of the sample A total of 672 mother-child pairs were included in the analysis. Around half of the infants had already been weaned before the end of their 12 months (51.9%). At 72 months of age, 15.6% were overweight and 12.9% were obese. A high prevalence of maternal overweight and/or obesity (52.4%) was also observed. Table 1 lists the characteristics of the sample by breastfeeding in its original continuous scale. Certain statistically significant differences between the two groups were detected: children who had been weaned before completing one year of life had mothers with lower educational levels, had higher family incomes, were more likely to have mothers who went out to work and engaged in more physical activity ( Table 1).

Stratification of children by breastfeeding or weaned at 12 months

As shown in Table 2, we observed that weaning at 12 months is an interaction factor that alters the association between mother going out to work and overweight/obesity. This explains the considerable difference in the prevalence ratios (PR) of overweight and/or obesity for the variable working mothers in the two strata (breastfed group PR 1.17; 95% CI 0.80-1.72; weaned group PR 1.83; 95% CI 1.28-2.50).

When associations between the three groups of characteristics (sociodemographic, genetic and children's characteristics) and the outcome were analyzed for the two strata,  certain factors were common to both groups (higher maternal educational level, higher family income, mothers with excess body weight, caesarean delivery and buying school meals), while working mothers and practicing sports outside of school were only associated with obesity among children who were weaned before 12 months (Table 2).

DISTAL MODEL SOCIOECONOMIC AND DEMOGRAPHIC CHARACTERISTICS

When the factors analyzed were adjusted using multivariate logistic regression analysis, it was found that having mothers with excess body weight was a predictor of risk of overweight/obesity at 6 years of age among children in both the strata. However, it is important to stress the fact that children breastfed for at least 12 months had a much lower risk (PR 1.92; 95% CI 1.15-3.24, Table 3) than those breastfed for shorter periods (less than 12 months) (PR 3.05; 95% CI 1.81-5.25, Table 4). In addition to maternal obesity, higher family income was a risk factor for overweight/obesity among breastfed children (Table 3). In contrast, for children who were weaned before 12 months, the predictors of overweight and/or obesity were as follows: higher maternal educational level, mother working outside the home at time of birth, and caesarean delivery (Table 4).

Discussion

Childhood obesity: a public health problem

The results of this study reveal a prevalence of overweight and obesity of 28.5% (15.6% overweight and 12.9% obesity), similar to figures reported in other studies [2,25,26]. These results show the growth of the problem as the children age and invite discussion on the need for monitoring speed of weight gain from the start of life. Controlling childhood obesity is a current challenge for public health worldwide.

Genetic and environmental aspects

The presence of obesity in adults is known to raise the risk of those adults having children who are also obese [2,10,27]. In this study, maternal obesity was associated with an increased risk of overweight/obesity, not only in breastfed children, but also in those who were weaned before 12 months.

Orera [28] found the risk children becoming obese increased 50% to 80% when the parents were obese. Marques-Lopes et al. [29] showed that the energy balance seemed to depend about 40% on genetic inheritance. It is    worth pointing out that there is an overlap between heredity and dietary habits and behaviors shared by parents and children in the family environment. Socioeconomic and demographic factors can act in parallel to the genesis of overweight and obesity in children and adults. In this study, some of these factors were associated with the outcome in both stratum of children studied. In those children breastfed for a longer period of time, lower family income was a protective factor. For those children who were weaned before 12 months, a lower level of mother's schooling was a protective factor and mother going out to work was a risk factor.

Similarly, Oliveira et al. [30], researching school-age children in the same city as our study, found the same association between overweight and/or obesity with parental income and educational level. Costa et al. [31] evaluated private and public schools and in a city in Southeast Brazil and concluded that the risk of obesity was greater in those with a more favorable socioeconomic status. Other authors found a high prevalence of overweight and obesity in middle-class children in Northeast Brazil [4].

The results of this study contradict existing evidence in the literature on developed countries [32], where a greater family income and higher educational level were protective against overweight/obesity. However, no clear relationship is reported between maternal education and socio-economic status and childhood obesity [33][34][35][36]. Further research is necessary to better understand and confirm this association, which may vary depending on social context.

There is a tendency for developing countries to import the lifestyle of more socioeconomically developed countries. Greater household availability of processed food products in Brazil is positively and independently associated with higher prevalence of excess weight and obesity in all age groups in a cross-sectional study [37]. It is also relevant to mention studies that have called attention to differences between the dietary habits of breastfed children and those who were not breastfed, showing that prematurely weaned children have less healthy diets [18], which can contribute to increasing the risk of obesity at later ages [13,27].

Female participation in the employment market is a result of the new domestic economics of modern society. The absence of mothers from the home environment may have an impact on children's nutritional status [38]. Some studies that have analyzed the associations between breastfeeding and overweight/obesity in children have discussed the increased risk of this outcome conferred on the children of mothers whose domestic roles have to compete with employment outside of the home [6,39]. Certain hypotheses can explain these results and are compatible with the results found in our study. Absence of mothers can alter children's dietary habits, reducing breastfeeding duration, encouraging premature introduction of complementary foods and making it more likely that sweets and treats will be given to children to compensate for spending the day without their mothers [6].

Caesarean delivery and childhood obesity

An interesting finding observed in this study was the association between type of delivery and overweight and/ or obesity among children weaned before 12 months. The sample studied exhibited a high prevalence of caesarean delivery (47.6%), around three times higher than the rates recommended by the WHO [40]. The risk of overweight and/or obesity was two times higher in the group of children born from a caesarean delivery (PR 1.98; 95% CI 1.14-3.50) than among those born by vaginal delivery. This association has so far been the subject of little discussion in the literature, but similar data have recently been reported in other studies. Goldani et al. [20] describe PR of obesity of 15.2% in adults aged 23 to 25 who were born by caesarean delivery (1.58; 95% CI: 1.23-2.02; p-value = 0.002). The authors discussed the biological plausibility of these results exploring the differences between the intestinal flora of children who did not have contact with the maternal vaginal flora during birth, delaying acquisition of bifidobacterium and leading to changes in the composition of the intestinal flora that are associated with excess weight later on. In parallel, other researchers have analyzed the profiles of faecal samples from infants, finding that the intestinal microbiota of children with greater weight than expected had a different bacterial composition, some of which were species associated with protection against obesity at later ages [41].

The association between intestinal microbiota and a predisposition to obesity is a recent subject in the literature. First described in experimental studies with rats, the "energy product" hypothesis suggests that energy expenditure may be increased by use of indigestible carbohydrates by enzymes produced by the microbiota or that    microbial metabolites exercise an influence on mechanisms that regulate their hosts intake and deposition of energy. However, the diversity of factors that influence the formation of microbiota, which include age, diet, lifestyle, ethnicity and genetics, combined with technical and methodological differences between intestinal flora analyses are challenges for studies that are ongoing. Currently, the question of whether changes to intestinal microbiota are a cause of obesity or a consequence of the disease remains unanswered [42]. Barros et al. [8] analyzed three Brazilian cohorts, but, after analyzing the prevalence of obesity in people aged 4, 11, 15 and 23 years who had been born by caesarean delivery, and after adjustment for confounding factors, the variable only had significance in one cohort and only for children aged 4. That study questions the role of alterations to intestinal flora as a cause of obesity, citing the lack of evidence to support the hypothesis and raising other possibilities, such as the presence of possible residual confounding factors; higher maternal educational level for example.
Income
It is interesting to mention that the literature has shown an increase in the risk of caesarean delivery in overweight or obese women, compared to those with a normal weight [43]. Moreover, caesarean delivery reduces breastfeeding in the first hour of life [44], which may complicate the management of lactation later on. Therefore, the influence of these factors on the association between caesarean delivery and childhood obesity should be considered.

Breastfeeding

Many studies have shown that prevention of diseases over the course of life is directly related to the nutrition received during gestation and the first years of life [9,10,13].

Breastfeeding has been seen as a protective factor against obesity, including some evidence showing that this protection increases with the time spent breastfeeding [6,[12][13][14][15][16]. On the other hand, some authors did not find this protective association between breastfeeding and obesity over the life course [45]. An interesting finding of De Jesus et al. [39], while researching the same sample as our study, found that exclusive breastfeeding in the first four months is protective against overweight at four years of age; however, during the construction of this study, the association between exclusive breastfeeding for the first four months or six months with overweight and obesity at six years was tested and there was no statistical significance between the different groups. These data may suggest that the protective factor of breastfeeding can be reduced over time due to other predictors such as genetics or lifestyle factors.

The finding that caesarean delivery loses statistically significance as a risk factor in the group of children breastfed for at least 12 months raises the hypothesis that even though the aggression (caesarean delivery) occurred during a susceptible period of development, the physiological and metabolic system was adjusted, by means of breast milk, reprogramming those infants who did not have contact with their mothers' vaginal microbiota during birth.

Final comments

This research suffers from the methodological limitations inherent to prospective studies, with their susceptibility to follow-up losses, which in this case totalled 12 children lost to follow-up from the 684 children estimated in the sample size calculation. On the other hand, some methodological limitations that can be observed in other studies were not repeated in this investigation, since data were collected prospectively during home visits in which the children's caregivers answered a 24-hour food recall. Additionally, the same technical team conducted all fieldwork from study outset to completion.

Conclusions

Maternal obesity and caesarean delivery were strongly associated with childhood overweight/obesity. A lower family income and lower level of education were identified as protective factors. Breastfeeding appears to modify the association between overweight/obesity and the predictors studied. This study highlights the necessity for further research to investigate the association between caesarean delivery and childhood obesity while considering the role of possible confounders and residual effect modifiers.

These investigations must consider the need to stratify analyses by type of feeding, since risk factors for obesity are different among breastfed children than among children who were not breastfed or were weaned prematurely.

Figure 1
1Diagram of hierarchical theoretical model of the determinants of overweight/obesity.

< 2 times minimum wage 0.41 (0.25 -0.67) <0.000 0.41 (0.25 -0.68) <0.000 0.50 (0.29 -0.85) 0.011 Mother overweight/obese ---------------------1.93 (1.17 -3.24) 0.011 1.92 (1.15 -3.24) 0.013

/obese ---------------------3.12 (1.86 -5.32) <0.000 3.05 (1.81 -5.25) <0.000 Caesarean delivery ------------------------------------------1.98 (1.14 -3.50) 0.016

Postnatal factorsPhysical activity at school Sedentary activities (>1h/day) Sport outside of school Plays ball games Rides a bicycle Buys school mealsOVERWEIGHT/OBESITYEducational level
Mother's age
Family income
Mother goes out to work at time of birth

INTERMEDIATE MODEL
GENETIC CHARACTERISTICS

Maternal BMI

PROXIMAL MODEL
CHILDREN'S CHARACTERISTICS
Neonatal factors
Type of delivery
Child sex
Birth weight
Gestational age

Table 1
1Description of the sample and comparison of characteristics, by breastfeeding using Student's t testVariable analyzed
N (%)
p-value

Mother's educational level

≤8 years of schooling
202 (30.1)
0.015

>8 years of schooling
470 (69.9)

Mother's age (years)

<20
104 (15.5)
0.692

≥20
568 (84.5)

Family income

<2 times minimum wage
321 (47.8)
<0.000

≥2 times minimum wage
351 (52.2)

Mother goes out to work at time of birth

Yes
241 (35.9)
0.021

No
431 (64.1)

Mother's BMI

Overweight/obesity
352 (52.4)
0.764

Underweight/normal weight
320 (47.6)

Type of delivery

Caesarean
327 (48.7)
0.123

Vaginal
345 (51.3)

Sex of child

Male
336 (50.0)
0.765

Female
336 (50.0)

Birth weight (grams)

<2,500
30 (4.5)
0.873

≥2,500
642 (95.5)

Gestational age

Preterm
26 (3.9)
0.620

Full term
646 (96.1)

Physical activity at school

Yes
179 (26.6)
0.035

No
486 (72.3)

Sedentary activities (>1 h/day)

Yes
621 (92.4)
0.369

No
51 (7.6)

Sport outside of school

Yes
79 (11.8)
0.091

No
593 (88.2)

Plays ball games

Yes
365 (54.3)
0.737

No
307 (45.7)

Rides a bicycle

Yes
468 (69.6)
0.939

No
204 (30.4)

Table 1
1Description of the sample and comparison of
characteristics, by breastfeeding using Student's t test
(Continued)

Buys school meals

Yes
250 (37.2)
0.113

No
415 (61.8)

N = number of children.
BMI = body mass index.
h = hour.

Table 2
2Bivariate analysis of factors associated with overweight/obesity stratified by breastfeeding at 12 months of ageVariables
Breastfeeding
Weaning
Interaction
p-value
N
Overweight/obesity
N
Overweight/obesity

n (%)
PR (95% CI)
n (%)
PR (95% CI)

Sociodemographic characteristics

Mother's educational level

≤8 years of schooling
120
20 (16.7)
0.57 (0.36 -0.89)
82
13 (15.9)
0.42 (0.25 -0.70)
0.291

>8 years of schooling
229
67 (29.3)
241
92 (38.2)

Mother's age (years)

<20
56
8 (14.3)
0.53 (0.27 -1.03)
48
11 (22.9)
0.67 (0.39 -1.16)
0.663

≥20
293
79 (27.0)
275
94 (34.2)

Family income (multiples of minimum wage)

<2
194
34 (17.5)
0.51 (0.35 -0.75)
127
31 (24.4)
0.65 (0.45 -0.92)
0.462

≥2
155
53 (34.2)
196
74 (37.8)

Mother working outside the home at time of birth

Yes
108
30 (27.8)
1.17 (0.80 -1.72)
133
59 (44.4)
1.83 (1.34 -2.51)
0.050

No
141
57 (23.7)
190
46 (24.2)

Genetic characteristics

Mother's BMI

Overweight/Obesity
167
56 (30.8)
1.66 (1.13 -2.45)
153
70 (41.2)
1.80 (1.28 -2.53)
0.591

Underweight/Normal weight
176
31 (18.6)
170
35 (22.9)

Characteristics of the children

Type of delivery

Caesarean
151
48 (31.8)
1.61 (1.12 -2.33)
176
75 (42.6)
2.09 (1.45 -3.00)
0.238

Vaginal
198
39 (19.7)
147
30 (20.4)

Sex of child

Male
165
37 (22.4)
0.83 (0.57 -1.19)
171
55 (32.2)
0.98 (0.71 -1.34)
0.520

Female
185
50 (27.2)
152
50 (32.9)

Birth weight (grams)

<2,500
17
3 (17.6)
0.70 (0.25 -1.98)
13
2 (15.4)
0.46 (0.13 -1.67)
0.588

≥2,500
332
84 (25.3)
310
103 (33.2)

Gestational age

Preterm
13
2 (15.4)
0.61 (0.17 -2.21)
13
1 (7.7)
0.23 (0.04 -1.52)
0.366

Full term
336
85 (25.3)
310
104 (33.5)

Table 2
2Bivariate analysis of factors associated with overweight/obesity stratified by breastfeeding at 12 months of age (Continued)Physical activity at school

Yes
78
26 (33.3)
1.46 (0.99 -2.15)
101
40 (39.6)
1.35 (0.98 -1.85)
0.825

No
268
61 (22.8)
218
64 (29.4)

Sedentary activities (>1 h/day)

Yes
320
80 (25.0)
1.04 (0.53 -2.03)
301
100 (33.2)
1.46 (0.67 -3.21)
0.488

No
29
7 (24.1)
23
5 (22.7)

Sport outside of school

Yes
31
11 (35.5)
1.49 (0.89 -2.48)
48
24 (50.0)
1.70 (1.21 -2.38)
0.539

No
318
76 (23.9)
175
81 (29.5)

Plays ball games

Yes
178
41 (23.0)
0.86 (0.59 -1.23)
187
57 (30.5)
0.86 (0.63 -1.18)
0.973

No
171
46 (26.9)
136
48 (35.3)

Rides a bicycle

Yes
242
59 (24.4)
0.93 (0.63 -1.37)
226
70 (31.0)
0.86 (0.62 -1.19)
0.714

No
107
28 (26.2)
97
35 (36.1)

Buys school meals

Yes
109
36 (33.0)
1.54 (1.07 -2.20)
141
55 (39.0)
1.42 (1.03 -1.94)
0.821

No
137
51 (21.5)
178
49 (27.5)

N = total number of children in "breastfeeding" or "weaning" group.
n = number of overweight and/or obesity children.
BMI = body mass index.
h = hour.

Table 3
3Logistic regression of characteristics associated with overweight/obesity among children breastfed for 12 monthsVariables
Model 1-distal
p
Model
2-intermediate

p
Model
3-proximal

p

PR (95% CI)
PR (95% CI)
PR (95% CI)

Table 4
4Logistic regression of characteristics associated with overweight/obesity among weaned childrenVariables
Model 1-distal
p
Model 2-intermediate
p
Model 3-proximal p

PR (95%CI)
PR (95% CI)
PR (95% CI)

Mother's educational level ≤8 years of schooling
0.39 (0.19 -0.74)
0.006 0.33 (0.16 -0.65)
0.002 0.39 (0.19 -0.78)
0.009

Mother working outside the home at time of birth 2.01 (1.22 -3.31)
0.006 2.34 (1.39 -3.98)
0.001 1.83 (1.05 -3.
AcknowledgementThis work was supported by grants from the Research Support Foundation of the State of Bahia (FAPESB).Competing interestsWe declare that there are no conflicts of personal or financial interests whatsoever between the results presented in this manuscript and any of the authors involved in the process of conducting this study.Authors' contributions DSP contributed to construction of the study design, analysis and interpretation of the data, and writing and revision of the manuscript. TOV worked on data collection, analysis and interpretation of the results, and writing and revision of the manuscript. SMAM contributed to analysis and interpretation of the data and revision of the manuscript. NFO contributed to construction of the study design and analysis and interpretation of the data. GOV worked on data collection, analysis and interpretation of the results, and writing and revision of the manuscript. All the authors read and approved the final manuscript.
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The inhibitory effect of Mesembryanthemum edule (L.) bolus essential oil on some pathogenic fungal isolates

Beauty E Omoruyi
Anthony J Afolayan
Graeme Bradley
The inhibitory effect of Mesembryanthemum edule (L.) bolus essential oil on some pathogenic fungal isolates
R E S E A R C H A R T I C L E Open Access
Background: Mesembryanthemum edule is a medicinal plant which has been indicated by Xhosa traditional healers in the treatment HIV associated diseases such as tuberculosis, dysentery, diabetic mellitus, laryngitis, mouth infections, ringworm eczema and vaginal infections. The investigation of the essential oil of this plant could help to verify the rationale behind the use of the plant as a cure for these illnesses.Methods: The essential oil from M. edule was analysed by GC/MS. Concentration ranging from 0.005 -5 mg/ml of the hydro-distilled essential oil was tested against some fungal strains, using micro-dilution method. The plant minimum inhibitory activity on the fungal strains was determined.Result: GC/MS analysis of the essential oil resulted in the identification of 28 compounds representing 99.99% of the total essential oil. A total amount of 10.6 and 36.61% constituents were obtained as monoterpenes and oxygenated monoterpenes. The amount of sesquiterpene hydrocarbons (3.58%) was low compared to the oxygenated sesquiterpenes with pick area of 9.28%. Total oil content of diterpenes and oxygenated diterpenes detected from the essential oil were 1.43% and 19.24%. The fatty acids and their methyl esters content present in the essential oil extract were found to be 19.25%. Antifungal activity of the essential oil extract tested against the pathogenic fungal, inhibited C. albican, C. krusei, C. rugosa, C. glabrata and C. neoformans with MICs range of 0.02-0.31 mg/ml. the activity of the essential oil was found competing with nystatin and amphotericin B used as control.Conclusion: Having accounted the profile chemical constituent found in M. edule oil and its important antifungal properties, we consider that its essential oil might be useful in pharmaceutical and food industry as natural antibiotic and food preservative.

Background

The global epidemics of HIV/AIDS appear to have stabilized in most regions. However, Sub-Saharan Africa remains heavily affected region according to the report of UNAIDS [1]. Among the Sub-Saharan Africa countries, South Africa happened to be the largest burden of HIV/ AIDS worldwide with an estimate of 5, 38 million out of 50.6 million indigenes in 2011 [2]. Majority of people living with HIV/AIDS are vulnerable to developing fungal illness because of their suppressed immune systems [3,4]. Fungal infections remain a significant cause of gastrointestinal disease, a consequence of HIV/AIDS contaminants especially in immunocompromised individuals [3,4]. The incidence of re-occurring fungal associated HIV/AIDS has increased dramatically. Candida albicans is one of the major causes of mucosal and bloodstream infections with over 85% to 95% cases reported if not treated [4]. Cryptococcus neoformans, a facultative organism that is very unique in attacking the lymphocytic cells, thereby creating a major gate way to HIV target. Meningitis, including lung infections are the common diseases related to C. neoformans [5].

Candida glabrata currently ranks the second or third causative agent of oral, vaginal, or urinary infections, which is often known as nosocomial disease. Its resistant mortality rates in compromised patients are very difficult to treat, especially with fluconazole drug [6]. Susceptibility of population with suppressed immunological defences against Candida rugosa infection in HIV/AIDS has emerged in spreading bovine mastitis in trauma patients [7,8]. Overall, Candida krusei ranked the fifth most common species that tends to be relatively seen in immunocompromised patients [8].

Over the years, the prevalence of fungal infection and its resistance to antibiotics drugs has brought to knowledge the importance to search for alternative treatments against infections [9]. It is noteworthy that researchers have directed their attention towards medicinal plants to develop better drugs against fungal infections. Traditional medicines have played an important role in health services around the globe, especially in South Africa due to wide arrays of phytochemicals with therapeutic properties [10]. Naturally, plants possesses free radical scavenging molecules, such as vitamins, terpenoids, phenolic acids, tannins, flavonoids, alkaloids, and other metabolites, which are rich in antioxidant with antimicrobial properties [11,12]. The ingestion of these natural antioxidants has shown to enhance the immune defence, reduce risks of cancer, cardiovascular disease, diabetes, and other diseases associated with ageing [12,13]. Owing to this fact, majority of South African population relies heavily on the use of plants and plant extracts for their well beings. Hence, much attention has been drawn to plantderived fungicides in recent years for the replacement of modern drugs [14]. Essential oil and their volatile constituents derived from medicinal plants have been reported to possess potent antifungal activities [15]. Majority of individuals who use essential oils from plant is less likely to contract infections disease [16]. Moreover, oils users who eventually contract an infectious disease trend to recover faster than those using antibiotics [17].

In South Africa, essential oils are usually used to preserve food against the growth of organisms. Thus many of these essential oils from medicinal plants are cheaply distributed and sold in the local market centers due to increased demands [2]. The high reliance on medicinal plants for health purposes necessitates the scientific validation of their therapeutic value and safety.

Mesembryanthemum edule (L.) bolus is an edible growing ground-cover plant commonly found in the costal districts of Eastern Cape of South Africa. The Xhosaspeaking people in this province usually administered alcohol, aqueous and essential oil extracts for the management of diseases common with HIV/AIDS infection [18]. Based on the ethnomedical information on this plant, the crude essential oil extract was screened for activity against Candida albican, C. rogusa, C. krusei, C. glabrata and C. neoformans. The activities of M. edule on mycobacteria causing tuberculosis (TB) have been described [14], but reports on the biological effect of its essential oil on pathogenic fungal isolated from HIV/ AIDS patients are limited. The aim of this research is to evaluate the inhibitory potential of M. edule essential oil against fungal isolated from HIV/AIDS patients. This study may justify its authentication to be used as complementary and alternative medicines.  [18].

Methods

Plant material

Essential oil

Volatile oil from the fresh leaves (500 g) was extracted for 3 h using a hydro-distiller (Clevenger's-type apparatus) in a 5-L round bottom flask fitted in a condenser. This process of extraction was repeated by another 500 g of the fresh leaves.

Gas chromatography-mass spectroscopy analysis

The essential oil extract was subjected to GC-MS analysis for identification of components in the department of Botany, University of Forth Hare. This was carried out using GC-MS (HP 6890) with a mass selective detector (HP5973). Identification of the components of essential oils was accomplished by comparison with the standards available in the database. The quantity of compounds was calculated by integrating the peak areas of spectrograms. A needle with the sample material (essential oils tested) was inserted directly into the inlet of a Hewlett Packard (HP 6890, USA) Gas Chromatograph. The temperature of the injection port was maintained at 220°C while the pressure at the inlet was maintained at 3.96 psi. A HP-5 MS (cross-linked 5% Phenyl Methyl Siloxane) column (30 m × 0.25 mm × 0.25 μm film thickness) was temperature-programmed from 60 to 150°C at 3°C min-1 after a 3 min delay. Helium was used as a carrier gas at 0.7 ml min-1. Mass spectra were recorded by a 5973 series Mass Selective Detector (MSD) [19].

Calculation of oil yield

Prior to the final extraction and obtaining the oil, a clean bottle of known mass was made available. At the end of extraction process, the essential oil obtained was carefully transferred into the bottle and the final mass noted.

The yield was obtained as follows: Mass of plant material distilled (g) = X; Mass of empty bottle (g) = A; Mass of bottle + oil extracted (g) = B; Mass of oil (g) = (B -A); Percentage (%) yield = [(B-A) ÷ X] 100 (Table 1). The essential oil was diluted in methanol (20% v/v) and a working concentration ranging between 0.005-5-mg/ml was used for the determination of Minimum Inhibitory Concentration (MIC).

Microorganisms and growth media

The fungi used in this study were chosen primarily on the basis of their importance as common pathogens of human infected with HIV/AIDS. Strains from the American type culture collection (ATCC) were used, including C. albicans ATCC 2091, C. krusei ATCC 204305, C. glabrata ATCC 2001, C. rugosa ATCC 10571 and Cryptococcus neoformans ATCC 66031. Both Sabouraud dextrose agar (SDA) and Sabouraud dextrose broth (SDB) were prepared according to the manufacturer's instructions. Each fungus was grown for 48 hour at 28°C in Sabouraud Dextrose Agar (Merck) plates. Scrape cell mass were transferred from each solid culture to 3 ml saline solution and then adjusted to 0.5 Mc Farland standard, which was confirmed by spectrophotometric reading at 580 nm [20]. Cell suspensions were finally diluted to 10 4 CFU/ml for the use in the assays.

Minimum Inhibitory Concentration (MIC)

The micro-dilution method using Sabouraud dextrose broth was employed to determine the minimum inhibitory concentration (MIC) of the plant extracts using 96 well microtitre plates. Firstly, an initially, 120 μl of sterile distilled water was added into each well of the first (A) and last (H) rows and also into all the wells of the last column (12). Secondly, 120 μl of SDB was added into each well of the second row (B) and 150 μl of same SDB was added into the remaining wells of the first column and then a 100 μl into the rest of the wells from the second column rightward. Fifty microlitre of the essential oil was then added into the third well of the first column, while 50 μl of the positive and negative control were separately added into the remaining wells of the first column. Following two-fold serial dilution method, each contents from the first column (starting from the third row) was mixed by transferring 100 μl into the second well of the same row and the procedure was repeated up to the 11th well of the same row and the last 100 μl from the 11th well was discarded. Hence various concentrations of the diluted essential oil ranging from 5 mg/ml to 0.005 mg/ml were prepared in the wells, following the two-fold dilution method. Thereafter, 20 μl of 0.5 Mc-Farland fungal suspensions was inoculated into the wells except those which contained sterile distilled water. Each treatment was performed in triplicates. The growth of the fungi was measured by determining the absorbance at 620 nm with a microtitre plate reader before and after incubation. Plates were incubated at 37°C for 24 hours. The lowest concentration which inhibited the growth of the fungi was considered as the minimum inhibitory concentration (MIC) of each extracts.

Statistical analysis

The antifungal experiments were made in triplicates and the data is reported as mean ± SD for (n = 1x3). Analysis of variance was performed by one way ANOVA using software statistical 5.5 (Stat Soft Inc, Tulsa, Ok). A probability value at P <0.05 was considered statistically significant.

Results and discussion

Percentage chemical compounds of the essential oil Hydro-distilled essential oil from fresh M. edule leaves analyzed by GC-MS resulted in the identification of 28 compounds representing 99.99% of the total essential oil. The essential oil was pale yellowish liquid with a fineagreeable characteristic aroma. The major compounds of the essential oil found based on their mass spectra peaks ( Figure 1) were the Tetra-decamethylcyclo-heptasiloxane with area peak of 23.81%, followed by Tetra-cosamethylcyclododecasiloxanes (22.51%), Octadecane (2.56%), Nephthalene (3.93%) and Eicosane (4.0%), Table 2.

The use of some of these chemical compounds has been well studied. Compounds such as Tetra-cosamethylcyclododecasiloxanes and Tetra-decamethylcyclo-heptasiloxane that ends with 'siloxanes' belongs to the wider class of organosilicon [21]. These compounds are made up of both organic and inorganic chemical compounds comprised of silicon, oxygen, carbon and hydrogen [22]. Siloxanes are commonly used in the cosmetic industries to produce deodorants, sunblocks, hairsprays and skincare [21]. In addition, siloxanes are an important product in the cook ware industry and kitchen utensils [21]. They are also used as effective industrial cleaning agents and in dry cleaning industries. In terms of properties, siloxanes are a good source of electric insulation, low chemical reactivity, low toxicity, high gas permeability, excellent resistance to oxygen, zone and UV light. Naphthalene is another chemical compounds derived from crude oil. It is a bicyclic aromatic hydrocarbon that is use as insecticide and as a repellent [23]. Majority of the volatile components analysed from plant essential oil largely belong to terpene. Terpenes are known to have strong biological activities and they are in involved in plant defences [24]. It has been well documented that the intake of terpenes can reduce accumulated toxins from the liver and kidneys in the body system [25]. In this study, Isoterpinolene (0.95%), Nephthalene (3.93%) and Bistrimethulesilyl N-acetyl (5.72%), were identified as monoterpenes respectively. Oxygenated monoterpene were found to be the highest (36.61%) constituents in the M. edule essential oil. Over the years, essential oil containing monoterpene hydrocarbons has offered a variety of healing properties, especially their ability to restore correct information in the DNA of a living cell and enhancement of other therapeutic components [26,27]. Isoterpinolene, one of the major monoterpenes observed in the study has been found capable of protecting human cells from free radical mediate oxidative stress [28]. It has been said that the oxygenated monoterpene compounds are more valuable than the monoterpene hydrocarbons due to their contribution to the fragrance of the essential oil [29].

Octadecanes (1.76%), 1-octadecane (0.80%) and Nonadecane (1.02%) were observed as sesquiterpene hydrocarbons in the M. edule essential oil. Essential oil containing sesquiterpenes have been used as therapeutic effect against inflammatory and allergic infections [30,31]. Research has found that people who consistently use sesquiterpenes essential oil have a higher level of resistance to illness than the average person [32]. Further indications revealed that if such individual eventually falls ill, he or she has a  tendency of recovering 60-70% faster than those who do not use essential oils [32,33]. Two Eicosane with a total area peaks of 1.43% were the major concentrated diterpenes detected from the M. edule essential oil. Oxygenated diterpene constituents accounted the third most concentrated hydrocarbons found in leaves, with a total essential oil content of 19.24%. Of these, Phytol content gave the highest amount with area peak of 12.41%, followed by all the Tetra-decamethylcycloheptasiloxanes, having area peak of 6.83%. Total amount of fatty acids and their methyl esters content present in the essential oil extract were found to be 19.25%. From Table 2 it is clear that benzoic acid represent the highest amount of 15.68% fatty acids of the essential oil.

Several bioactive compounds have been isolated from M. edule, such as phenolics, flavonoids, proanthocyanidins, alkaloids, saponins, tannin, rutin, cactichin, ferulic acid hyperoside, oleanolic acid, catechin and epicatechin [34][35][36]. Unfortunately, there is no available information on the profile chemical constituents from M. edule leaf essential oil. The different phytochemical constituents of monoterpene, sesquiterpenes, diterpenes and fatty acid esters have been reported to have antioxidant, antimicrobial, immune-modulating activities [37][38][39].

Microbiological screening of essential oil from many plants (Pimpinella anisum L.  [40][41][42]. From the antifungal results presented in Table 3, the MIC of the essential oil effectively inhibited the growth of the organisms when compared to nystatin and amphotericin B used as control. The extent of inhibition on the fungal growth is dependent on the concentration used. Candida albican and C. krusei which are the most dangerous organisms in human system had the most sensitive treatment of 0.02 and 0.04 mg/ml activity.

These findings agree with studies done on the Candida strain isolated from infants and standard strains [43]. Both strains were greatly inhibited by 80.95% and 14.23% essential oil of thyme, pennyroyal and lemon [43].

Minimum inhibitory activity of the essential oil against C. rugosa and C. neoformans (0.08 mg/ml) was significantly different from that of C. glabrata at 0.31 mg/ml. Antifungal activity of Lavandula viridis L. essential oil against Cryptococcus neoformans was 0.64 μl/ml, which is significantly higher than our result [44]. Other observations from Saeid and Seddighe [43], reported 2.3% activity of essential oil against C. glabrata.

Conclusion

Conclusively, the results obtained from the GC-MS resulted in the identification of 28 hydrocarbons of the total essential oil. The phytoconstituent present in the essential oil are in the family of monoterpenes, sesquiterpenes, diterpenes, and fatty acids esters. Oxygenated monoterpenes occupies the major constituents of the oil, followed by fatty acids and oxygenated diterpenes. The therapeutic potency of M. edule used as traditional medicine thus contains properties that inhibit the growth of fungi activity. The growth of Candida albican and C. krusei which are the most common agent in candiadiasis patients were greatly reduced by M. edule essential oil. Mesembryanthemum edule match a candidate species for future studies on novel and alternative remedy for the treatment of microorganism infections. However, further studies will be carried out to isolate and identify the active compounds, and to determine their exact mechanism of action.

After obtaining the human ethics certificate (BRA0S1O-MUO1) approved by the University of Fort Hare's research ethics committee, the survey of this medicinal plant was carried out in June 2012, fresh leaves of M. edule were supplied by herbalist from Nkonkobe Municipality. The taxonomical identity of the plant was confirmed by a botanist Prof. DS Grierson and a voucher specimen was kept in the Griffin Herbarium of the Botany Department, University of Fort Hare as (Omo 2011/1-Omo 2011/19)

Figure 1
1A typical Gas chromatography profile showing the chemical analysis of M. edule essential oil.

(anised), Syzygium aromaticum L. (clove), Cuminum cyminum L. (cumin), Allium sativum L. (garlic), Laurus nobilis L. (laurel), Citrus sinensis (L.) Osbeck (orange sweet), and Origanum vulgare L. (oregano), Tulbaghia violacea Harv L.F and Eucalyptus grandis W.Hill ex Maiden) have earlier been studied to have high antibacterial, antifungal, antiviral, antiparasitic and antidermatophytic properties

Abbreviations HIV: Human immunodeficiency virus; AIDS: Acquired immunodeficiency syndrome; M. edule: Mesembryanthemum edule; UANOVA: Analysis of variance; MSD: Mass Selective Detector; GC-MS: Gas chromatography-mass spectrometry; ATCC: America type culture collection; MIC: Minimum inhibitory concentration.

Table 1
1Percentage yield essential oil from M. edule leavesItem
Essential oil of leaf

Percentage yield
4.21%

Colour
A very pale yellow

Solubility in methanol 20%
1 in 1/V

Table 2
2Compounds obtained from GC/MS analysis of M. edule leaf part essential oil Percentage composition of M. edule essential oil analysed by GC/MSPK/No
Compounds
Kovats index
Peak %
Chemical formula

Monoterpenes
10.6

1
Isoterpinolene
1429
0.95
C 10 H 16

2
Nephthalene, 1,2-dihydro-2,5,8-tri
1546
2.03
C 12 H 10

3
Nephthalene, 1,2-dihydro-2,5,8-tri
1548
1.90
C 12 H 10

4
Bistrimethylsilyl N-acetyl EICOSAS
1978
5.72
C 15 H 33 NO 5 Si 3

Oxygenated monoterpenes
36.61

5
Mercaptoacetic acid, bis (trismethylsilyl)
3740
2.07
C 8 H 20 O 2 SSi 2

6
Eicosamethylcyclodecasiloxane
1936
2.58
C 8 H 24 O 4 Si 4

7
N-Octanol
1297
1.59
C 8 H 18 O

8
Nonylaldehyde
1345
2.29
C 9 H 18 O

9
Trans-β-demascenone
1538
3.42
C 13 H 18 O

10
Trans-2-tridecenal
1406
0.85
C 13 H 24 O

11
Tetradecamethylcycloheptasiloxane
1627
7.39
C 14 H 42 O 7 Si 7

12
Tetradecamethylcycloheptasiloxane
1646
13.57
C 14 H 42 O 7 Si 7

13
Tetradecamethylcycloheptasiloxane
1654
2.85
C 14 H 42 O 7 Si 7

Sesquiterpenes
3.58

14
Octadecane
1991
0.64
C 18 H 38

15
Octadecane
2092
1.12
C 18 H 38

16
1-octadecene
2266
0.80
C 18 H 36

17
Nonadecane
2284
1.02
C 18 H 40

Oxygenated sesquiterpene
9.28

18
2-pentadecanone,6,10,14-trimethyl
2014
9.28
C 18 H 36 O

Diterpenes
1.43

19
Eicosane
2215
0.65
C 20 H 42

20
Eicosane
2439
0.78
C 20 H 42

Oxygenated diterpenes
19.24

21
Phytol (2-Hexadecen-1-o1, 3,7,11,15-tetramethyl)
2289
12.41
C 20 H 40 O

22
Trisiloxane,1,1,1,5,5,5-hexamethyl-3-[(trimethylsilyl)oxy]
(Tetracosamethylcyclododecasiloxane)

2302
1.64
C 24 H 72 O 12 Si1 2

23
Tetrasiloxane,1,1,1,5,7,7,7-heptamethyl-3, bis[(trimethylsilyl)oxy]
(Tetracosamethylcyclododecasiloxane)

2420
1.66
2 C 24 H 72 O 12 Si1 2

24
3-Isopropoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tri(trismethylsiloxy)
tetrasiloxane (Tetracosamethylcyclododecasiloxane)

2538
1.69
2 C 24 H 72 O 12 Si1 2

25
Tetrasiloxane-1,1,1,5,7,7,7-heptamethyl-3,3 bis[(trismethylsilyl)oxy)]
(Tetracosamethylcyclododecasiloxane)

2680
1.84
2 C 24 H 72 O 12 Si1 2

Fatty acids
19.25

26
Benzoic acid, 2,5-bis (trimethylsiloxy-,trimethylsilyl ester
(Tetracosamethylcyclododecasiloxane)

1841
15.68
C 16 H 30 O 4 Si 3

27
Hexadecanoic acid, ethyl ester
2183
0.89
C 18 H 36 O 2

28
Hexadecanoic acid, 1-methylethyle ester
2215
2.68
C 19 H 38 O 2

Total compounds (%)
99.99

Bold data (10.6; 36.61; 3.58; 9.28; 1.43; 19.24; 19.25; 99.99) represents the percentage peak area values calculated for each terpenes and fatty acids
hydrocarbon.

Table 3
3Minimum inhibitory concentration (MIC) of the extracts against the five fungal abc Values within rows are significantly different (p > 0.05). The minus sign (−) indicate negative result.Minimum inhibitory concentration

Test organisms
Essential oil
(mg/ml)

Nystatin
(mg/ml)

Amphotericin B
(mg/ml)

C. albican
0.02
0.02
0.02

C. krusei
0.04 a
0.02 b
0.009 c

C. rugosa
0.08 a
0.02 b
0.009 c

C. glabrata
0.31 a
0.04 b
0.02 b

C. neoformans
0.08 a
0.009 b
0.02 c

http://www.biomedcentral.com/1472-6882/14/168
AcknowledgementThis work was supported by the National Research Foundation of South Africa, University of Fort Hare.Competing interestsThe authors declare that they have no competing interests.Authors' contributions BEO was responsible for the collection of plant materials from the traditional healers, carried out 90% of the experiments, and drafted the manuscript. GB edited the manuscript. AJA participated in the GC-MS analysis, supervised in the laboratory assay and made substantial contribution to revise the manuscript critically. All authors read and approved the final manuscript.
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The inhibitory effect of Mesembryanthemum edule (L.) bolus essential oil on some pathogenic fungal isolates. Omoruyi, BMC Complementary and Alternative Medicine. 14168Cite this article asCite this article as: Omoruyi et al.: The inhibitory effect of Mesembryanthemum edule (L.) bolus essential oil on some pathogenic fungal isolates. BMC Complementary and Alternative Medicine 2014 14:168.