Annona montana Fruit and Leaves Improve the Glycemic and Lipid Profiles of Wistar Rats

Abstract

Species of the family Annonaceae has been used traditionally as a medicinal plant in tropical regions of South and North America and in Africa. Annona montana is known popularly as false graviola and originates from tropical America and can be cultivated throughout Brazil. There are no studies in the literature that associate A. montana with the metabolic profile of animals. Therefore, the purpose of this work was to assess the effects of the consumption of pulp and leaves of this plant on the metabolic profile of Wistar rats. The animals, which were treated for 40 days, were divided into two control groups—treated with water via gavage and ad libitum, respectively, and two treated groups—one treated with leaf juice and the other with pulp juice of the fruit. Glycemia, lipids, and body weight were found to decrease and high density lipoprotein-cholesterol (HDL-c) levels to increase in the animals treated with leaf juice. The group treated with pulp juice showed a reduction in lipids and augmented HDL-c. The use of A. montana may have beneficial effects in the prevention of diabetes mellitus and dyslipidemia and may thus contribute to the prevention of cardiovascular diseases.

Introduction

I n Brazil, there are 26 genera of the family Annonaceae and approximately 260 species, but only a few of them produce edible fruits; several are wild and some have been domesticated. Annona muricata, one of the most well-known species, has been used traditionally as a medicinal plant in tropical regions of South and North America and in Africa. All the parts of the plant can be used for therapeutic purposes.^1,2

A. montana is known popularly as guanabana or false graviola due to its similarity with graviola (A. muricata). It originates from tropical America and can be cultivated throughout Brazil. Its fruit is oval in shape, with a diameter of 10–15 cm and a length of up to 25 cm; its rind is green and composed of tiny spicules. The pulp may be white or yellowish, with a soft texture, and its seeds are brownish-yellow.^3,4

In addition to A. muricata, other species of the genus (such as A. crassiflora, A. diversifolia, and A. squamosa) are used in some countries, with allegedly medicinal properties ranging from anticancer, antiparasitic, antispasmodic, antidiarrheal, antiulcer, sedative, analgesic (antinociceptive activity), hypotensive, and vermifugal effects to skin diseases.^2,5
–7

On the other hand, increasing global industrialization has led to profound changes in the lifestyle of the world's populations, giving rise to rising indices of chronic degenerative diseases such as diabetes mellitus, metabolic syndrome, and cardiovascular diseases, which are among the principal causes of death and which have been increasingly affecting younger populations. Many plants have been used for the purpose of reducing risk factors associated with the occurrence of these disorders.^8

–15

Several authors have isolated different phytochemicals (such as acetogenins and montanacins) from distinct parts of A. montana. These compounds may have significant effects on the control of risk factors for diabetes and cardiovascular diseases.^16
–18

In view of the ongoing efforts to identify the real effects of so-called medicinal plants, and considering the increasing distribution of Annonaceae species in Brazil, as well as the lack of studies that associate A. montana with the metabolic profile, the purpose of this work was to assess the effects of the consumption of pulp and leaves of this plant on the biochemical profile of Wistar rats.

Materials and Methods

Groups of animals

This study was approved by the Animal Research Ethics Committee of the University of Marília (UNIMAR). The animals were treated according to the Guide for the Care and Use of Experimental Animals (which sets down the principles for the care of laboratory animals).

Forty male Wistar rats were used, weighing approximately 250 g, which were kept in the vivarium at UNIMAR (University of Marília) under a dark/light cycle of 12 hours, room temperature of 22±2°C, and relative air humidity of 60±5%. Throughout the experiment, the animals of the treated groups were fed A. montana leaf juice and pulp juice and rat food ad libitum.

After a period of 7 days of acclimation to the laboratory, the animals were divided randomly into four groups (n=10) treated for 40 days, as follows: G1 received water ad libitum; G2 received A. montana leaf juice ad libitum; G3 received water via gavage; and G4 was gavage-fed with pulp juice (the pulp juice was gavage-fed, because it is highly viscous and would otherwise have clogged the rodent drinking valves).

Weight gain was monitored once a week on days 1, 8, 15, 23, 31, and 39 of the experiment. The animals were fed daily, and their consumption (of rat food and water) was recorded based on the leftovers found each day.

Collection of plant material

The species used in this study was collected in Marília, state of São Paulo, Brazil, and was identified at the Ribeirão Preto School of Philosophy, Sciences and Literature of the University of São Paulo. Determined/confirmed by: M. Groppo X.2011. Collector and number: M. Groppo 2077. Registered in the Herbarium under no. SPFR 13139.

Preparation of A. montana leaf juice

The juice was prepared from A. montana leaves by grinding them with water in a blender, in a proportion of 1:5 (leaves:water). The juice was filtered through gauze and stored in 500-mL plastic bottles at freezing temperature (−18°C).

Preparation of A. montana pulp juice

The pulp juice was prepared with ripe fruits which were beaten with water in a blender, without removing the seeds, in a proportion of 1:5 (pulp:water). The juice was filtered through gauze and stored in 20-mL amber jars at freezing temperature (−18°C). On each day of the treatment, a jar was removed from the freezer and left to stand at room temperature until it liquefied. This juice was administered via gavage to each animal, 0.5 mL in the morning and 0.5 mL in the afternoon.

Collection of blood samples and determination of the biochemical profile

After 40 days of treatment, the animals were anesthetized with Hypnol^® (sodium pentobarbital) until complete sedation, after which blood samples were drawn to determine their biochemical profile: glycemia, aspartate transaminase (AST), alanine aminotransferase (ALT), total cholesterol, high density lipoprotein-cholesterol (HDL-c), and triacylglycerides. The glucose and lipid levels were measured in mg/dL, and AST and ALT in U/L.

The exams were performed at the Clinical Analysis Lab of the University Hospital of UNIMAR (Laboratório São Francisco), and the results were interpreted according to the American Diabetes Association.¹⁹

Statistical analysis

The variables are presented as means and standard deviations. The data were analyzed by the Student's t-test with a 5% level of significance.

Results

The results indicated that there was no variation in the biochemical profile between the control groups (G1 and G3) (Table 1).

Table 1.

Biochemical Parameters of the Animals of the Control Groups (G1 and G3)

	Groups
	G1	G3	P value
Glycemia (mg/dL)	163.20±13.74	169.90±32.24	.0667
Triacylglycerides (mg/dL)	104.90±18.28	109.00±9.58	.0996
Total cholesterol (mg/dL)	80.00±7.18	78.80±8.48	.1456
HDL-c (mg/dL)	17.10±3.20	21.50±1.24	.0576
AST (U/L)	133.00±15.19	112.60±19.03	.0778
ALT (U/L)	76.10±16.57	58.10±19.73	.1298

Mean±SD. G1 is the control group that received water ad libitum, and G3 is the control group that received water via gavage.

HDL-c, high density lipoprotein-cholesterol; AST, aspartate transaminase; ALT, alanine aminotransferase; SD, standard deviation.

As can be seen in Table 2, the values of glycemia and plasma lipids declined significantly and the HDL-c values increased considerably after the treatment with A. montana leaf juice (G2) when compared with the control group (G1). No significant variations were found in the levels of hepatic enzymes (AST and ALT).

Table 2.

Biochemical Parameters of the Animals of G1 and G2

	Groups
	G1	G2	P value
Glycemia (mg/dL)	163.20±13.74	130.50±9.40	.00000
Triglycerides (mg/dL)	104.90±18.28	57.50±6.38	.00000
Total cholesterol (mg/dL)	80.00±7.18	51.40±4.95	.00000
HDL-c (mg/dL)	17.10±3.20	29.70±1.64	.00000
AST (U/L)	133.00±15.19	127.40±10.29	.1402
ALT (U/L)	76.10±16.57	72.50±7.46	.26942

Mean±SD. G1 is the control group that received water ad libitum, and G2 is the group treated with Annona montana leaf juice.

The animals treated with A. montana pulp juice (G4) (Table 3) showed a significant decline in the values of triglycerides and total cholesterol and a substantial increase in HDL-c values when compared with the control group (G3). No significant difference was observed in glycemic and ALT levels.

Table 3.

Biochemical Parameters of the Animals of G3 and G4

	Groups
	G3	G4	P value
Glycemia (mg/dL)	169.90±32.24	160.00±27.24	.0814
Triacylglycerides (mg/dL)	109.00±9.58	62.33±9.81	.0041
Total cholesterol (mg/dL)	78.80±8.48	63.56±8.40	.0187
HDL-c (mg/dL)	21.50±1.24	25.89±2.47	.0356
AST (U/L)	112.60±19.03	98.44±21.42	.0024
ALT (U/L)	58.10±19.73	67.11±12.34	.0768

Mean±SD. G3 is the control group gavage-fed with water, and G4 is the group gavage-fed with A. montana pulp juice.

In Table 4, a comparison of the treated groups indicates that the values of glycemia, total cholesterol, AST, and ALT are significantly lower in the group treated with A. montana leaves (G2) than in the one treated with the fruit's pulp juice (G4).

Table 4.

Biochemical Parameters of the Animals of G2 and G4

	Groups
	G2	G4	P value
Glycemia (mg/dL)	130.50±9.40	160.00±27.24	.0456
Triglycerides (mg/dL)	57.50±6.38	62.33±9.81	.1077
Total cholesterol (mg/dL)	51.40±4.95	63.56±8.40	.0478
HDL-c (mg/dL)	29.70±1.64	25.89±2.47	.0480
AST (U/L)	127.40±10.29	98.44±21.42	.0056
ALT (U/L)	72.50±7.46	67.11±12.34	.1298

Mean±SD. G2 is the group treated with A. montana leaf juice ad libitum, and G4 is the group gavage-fed with A. montana pulp juice.

Table 5 indicates that all the groups (G1–G4) underwent significant changes in weight from the beginning to the end of the treatment. However, the treated groups (G2 and G4) presented a numerically lower weight gain than the animals that did not consume the plant.

Table 5.

Weights (in g) of the Animals of the Control and Treated Groups

	Time
Groups	Beginning	End	P value
G1	258.91±38.91	339.11±43.22	.0421
G2	250.35±44.4	301.44±18.29	.0389
G3	260.2±25.60	356.25±15.90	.0054
G4	248.37±36.07	323.34±29.36	.0008

Mean±SD. Weight of the animals of the control groups (G1 and G3) and the groups treated with A. montana leaf juice (G2) and pulp juice (G4) at the beginning and end of the treatment.

Table 6 shows that there is no significant difference between the body weight of the control groups (G1 and G3) at the end of the treatment. The group treated with A. montana leaves (G2) showed a significant decline in weight gain when compared with the control (G1). The group treated with pulp juice (G4) did not show a significant difference in relation to the control group (G3). Moreover, no significant variation was found in a comparison of the two treated groups (G2 and G4).

Table 6.

Comparison of Body Weights (in g) of the Groups of Animals at the End of the Treatment

Groups			P value
G1×G2	339.11±43.22	301.44±18.29	.0222
G1×G3	339.11±43.22	356.2±15.90	.0675
G2×G4	301.44±18.29	323.34±29.36	.3345
G3×G4	356.25±15.90	323.34±29.36	.0798

Mean±SD. Comparison of the body weight of the controls (G1 and G3) and the groups treated with A. montana leaf juice (G2) and pulp juice (G4) at the end of the treatment.

Discussion

Metabolic syndrome involves several risk factors related to insulin resistance, diabetes mellitus, and cardiovascular diseases. Among these factors are obesity, hyperglycemia, hypertriglyceridemia, low HDL-c levels, and arterial hypertension. Many studies have demonstrated that the use of medicinal plants and functional foods can reduce the occurrence of these risk factors.^13,14,20 The results of this work corroborate those studies and demonstrate that the use of A. montana may be beneficial for the control and prevention of the aforementioned risk factors, since it promoted a reduction in glycemia, total cholesterol and triglyceride levels, and augmented HDL-c levels.

In the stem parts of A. montana, Wu et al. ¹⁶ detected the presence of several components (N-trans-feruloyltyramine, N-p-coumaroyltyramine, and N-trans-caffeoyltyramine, lignans, syringaresinol, an aromatic aldehyde, syringaldehyde, beta-sitosterol, and beta-sitosterol-beta-d-glucoside), which exhibited an antiplatelet aggregation activity. Wang et al. ^17,18 isolated four novel mono-tetrahydrofuran acetogenins, montanacins, from the ethanol extract of A. montana leaves. Chuang et al. ²¹ analyzed the extract of A. montana seeds and found new cyclopeptides, cyclomontanins A–D, annomuricatin C, and (+)-corytuberine, which exhibited an in vitro anti-inflammatory activity. Alvarez Colom et al. ²² isolated 10 acetogenins from A. montana leaves, including tucupentol, annonacin-A, cis-annonacin-10-one, aromine, and gigantetronenin. The occurrence of these phytochemicals in the plant is related with modifications in animal metabolism; hence, the presence of these compounds in the species under study may have contributed to improve the metabolic profile of the animals treated with A. montana. Jossang et al. ²³ isolated an acetogenin from A. montana seeds and named it annomontacin. They observed cytotoxic effects of this compound on cancer. Mootoo et al. ⁴ found three new acetogenins (anmontanins A–C) from the leaves of A. montana. They also isolated murisolin and annonacin (known acetogenins) from the seeds. Liaw et al. ²⁴ found nine new acetogenins from the seeds of A. montana (montalicins A–E, cis-annoreticuin, montalicins F, I, and J, along with eight known acetogenins 10–17). These new acetogenins showed selectively potent cytotoxicity against two human cancer cell lines.

The effects of other species of Annona have also been evaluated. A study by Adewole and Ojewole² found that an aqueous extract of A. muricata leaves exerted hypoglycemic, hypolipidemic, and antioxidant effects on diabetic rats. These effects may be attributed to the presence of compounds such as tanins, polyphenols, flavonoids, steroids, saponins, and a series of secondary metabolites that can act in the reduction of glycemia as well as provide hypolipidemic, hypotensive, and anti-inflammatory effects.^25
–27 Adeyemi et al. ²⁸ used a methanol extract of A. muricata on diabetic rats and also observed hypoglycemic effects. Other study shows that Streptozotocin-induced diabetic rats treated with methanolic extracts of A. muricata leaves presented regeneration of the beta-cells of islets of pancreatic islet.²⁹

Baskar, et al. ³⁰ evaluated the antioxidant potential of A. muricata, A. squamosa, and Annona reticulata and concluded that A. muricata has stronger antioxidant effects in vitro, which could increase the range of therapeutic effects of this species.

Gupta et al. ³¹ used an extract of A. squamosa on diabetic rats and rabbits and observed hypoglycemic and hypolipidemic effects, as well as augmented HDL-c levels. Kaleem et al. ³² used the same species, also on diabetic rats, and also reported beneficial effects on lipemia, glycemia, and insulin and antioxidant enzyme levels, concluding that it may be useful in the prevention of both early and long-term complications caused by diabetes, such as lipid peroxidation. Gupta et al. ³³ evaluated the effects of an A. squamosa extract and reported significantly diminished triglycerides and total cholesterol and an augmented antioxidant enzyme activity (catalase, superoxide dismutase, glutathione reductase, and reduced glutathione) in different tissues.

Dragano et al. ⁶ studied A. crassiflora (also known as marolo), which is a well-known plant in folk medicine. However, these authors did not detect any decline in the levels of glucose, total cholesterol, and triglycerides in mice treated with the fruit's pulp.

According to their chemical nature, phenolic compounds act as reducing agents that interrupt the oxidation chain, establishing free radicals through electron or hydrogen donation. Numerous studies have demonstrated that the antioxidant power of these compounds has effects on the prevention de cardiovascular diseases.^34
–36

Khallouki et al. ³⁷ used a methanol extract of the root of Annona cuneata and found different polyphenolic components with a high antioxidant potential (such a squalene, hydroxybenzaldehyde, vanillin, vanillic acid, tyrosol, 3,4 dihydroxybenzaldehyde, and p-hydroxybenzoic acid), which explain why this plant is frequently used in folk medicine in Congo to treat different illnesses. Yeo et al. ³⁸ demonstrated that an ethanol extract of Annona senegalensis diminished the number of inflammatory cells, which also suggests minimization of vascular damage.

Agostini et al. ¹ studied the chemical characteristics of Annona coriaceae and described, among other compounds, the presence of fibers, and vitamin C and A. If these compounds are also present in the species used in this work, they may also have contributed to improve the biochemical profile of the studied animals.³⁹

The use of the leaf and pulp juice of A. montana did not promote changes exceeding the reference values in the hepatic enzymes of the Wistar rats in this study, indicating its possible use in the dosages that were administered to the animals. From their study of the use of A. muricata leaves, Adewole and Ojewole² concluded that the leaf extract of this plant was only moderately safe in the experimental model they used (Mus domesticus).

Conclusions

The results of this work demonstrate that the use of A. montana fruit, as well as its leaves, which are normally considered residue, may have beneficial effects in the organism of the animals under study, and may therefore be beneficial to health, helping to lower the incidence of chronic degenerative diseases.

In view of these findings, we suggest that further studies be conducted to identify the compounds present in this plant, elucidate their effects in humans, and establish ideal and safe doses of consumption to ensure the effectiveness of the benefits of this plant.

Footnotes

Acknowledgment

The authors are indebted to M. Groppo from Ribeirão Preto School of Philosophy, Sciences, and Literature of the University of São Paulo for the identification of the plant used in this work.

Author Disclosure Statement

All the authors report no conflicts of interest.

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