Effect of Prosthechea karwinskii (Orchidaceae) on obesity and dyslipidemia in Wistar rats

Peer review under responsibility of Alexandria University Faculty of Medicine.

Available online 2 January 2017

Abstract

Background

Prosthechea karwinskii is an endemic Mexican orchid, it’s currently used as decorative element and in the traditional medicine to treat diabetes and some problems related to inflammatory processes.

Aim

To determine antioxidant activity index (AAI) and to validate by the first time and through an rat model the hydroalcoholic extract obtained from Prosthechea karwinskii, a plant used in traditional medicine for treat conditions relate to the metabolic syndrome.

Methods

For in vivo assays 25 weaned male Wistar rats were divided into a control group (CG; n = 5) and a Metabolic Syndrome group (MS; n = 20). The rats of the latter were induced to MS with 40% sucrose in the drink water during 13 weeks. After MS induction this group was subdivided into 4 groups: MS group (n = 5) received sucrose, and three groups receiving 200 mg/kg of body weight of each extract pseudobulb (P, n = 5), leaf (L, n = 5), and flower (F, n = 5). All treatments were followed for 13 days. Blood was collected at the end of the study to measure glucose, cholesterol and triglycerides. AAI were measured in the extracts by the method of DPPH. The results were analyzed using MINITAB 16.1.0, and the statistical significance was determined by ANOVA and a Tukey’s test (P < 0.05).

Results

Leaves (L) extract had highest values in AAI, followed by flowers (F) and pseudobulb (P) extracts. Leaves extract had highest reducing effect on glucose level, while flower extract had highest reducing effect on the cholesterol and triglycerides levels.

Conclusions

The P. karwinskii extracts evaluated here reduces the glycemic and lipidemic parameters in Wistar rats with MS induced. These effects may be attributed to the high antioxidant capacity of the extracts.

Keywords:

• Antioxidant activity
• Cholesterol
• Diabetes
• Glucose
• Medicinal plants
• Triglycerides

1 Introduction

Medicinal plants play an important role in the introduction of new therapeutic agents as a source of biologically active substances with antihyperlipidemic and antihyperglycemic properties, among other effects.¹ Hyperglycemia results in an increased oxidative stress due to reduced endogenous antioxidants in the body² and imbalance in adipose tissue, influencing lipid regulation and triggering cardiovascular disease. Metabolic syndrome (MS) is the term used to designate a set of interrelated conditions that include hyperglycemia, hyperlipidemia, obesity and hypertension.^3–6 In traditional medicine, plants with biological activity affecting metabolic disturbances related to the pathophysiology of MS have been evaluated, including species from Orchidaceae family. Asian orchids such as Nervilia plicata (Andrews) Schltr.,⁷ Dendrobium chrysotoxum Lindl.⁸ and Dendrobium denneanum Kerr.,⁹ have shown to have hypoglycemic effects. In Mexico some orchid species have also been evaluated for conditions related to MS; Scaphyglottis fasciculate Hook. for its potential relaxing effect on cardiac contractions,¹⁰ Laelia autumnalis (La Llave & Lex.) Lindl. for its antihypertensive effect,¹¹ Laelia anceps Lindl. for its antihypertensive and vasorelaxant effects,¹² and Prosthechea michuacana (La Llave & Lex.) W.E. Higgins for its hypoglycemic activity.¹³

An endemic orchid to the mountains of southern Mexico that is used in the traditional medicine is Prosthechea karwinskii (Mart.) J.M.H. Shaw. Different parts of this orchid are used as remedy to treat hyperglycemia (pseudobulb and leaves), cough (pseudobulb and flowers), burns (pseudobulb), and to prevent miscarriages (flowers).¹⁴ The flowering of this species coincides with Easter celebrations and therefore this orchid is also used in religious decorations at homes and churches.^14–16 A previous phytochemical study¹⁷ of our research group has identified the presence of phenolic constituents in this orchid, like tyrosol, apigenin-7-glucoside, caffeic acid, vainillin, p-cumaric acid and ferulic acid. These compounds are known by their cardioprotective effects^18,19 due to its ability to inhibit cholesterol oxidation. Furthermore, phenolic compounds can inhibit adipose tissue growth due to their antiangiogenic activity and their ability to regulate adipocyte metabolism.²⁰

The goal of this research was to determine the antioxidant activity and to validate, by the first time, el traditional use of Prosthechea karwinskii in the treatment of some conditions relate to the metabolic syndrome, for which some parts of the plant are employed (pseudobulb, leaf, or flower). For this, the plant extracts were obtained with ethanol-deionized water and then were evaluated in a rat model.

2 Materials and methods

2.1 Plant material and extracts

Plant material was collected in 2012 and 2013 from specimens that were used as Easter decorations in Villa de Zaachila, Oaxaca. Additional material was collected in San Pedro and San Pablo Teposcolula with the permission of the local authority. Taxonomic determination of the plant was done and pressed specimens were prepared and deposited in OAX Herbarium of the Instituto Politécnico Nacional (Solano 4037). Since in traditional medicine is known that each part of this orchid is used separately for particular condition¹⁴, the plant material was separated into pseudobulbs, leaves and flowers; each portion was dried, pulverized, and stored at room temperature (R.T.) until use. Ten grams of each dried part were placed in 400 ml of ethanol-deionized water solution (1:1, w/v) for 7 days at R.T., frequently stirred, then filtered and concentrated by solvent evaporation at R.T. The yield of the extraction for each plant part was: 18.8% for pseudobulbs, 16.8% for leaves, and 33.6% for flower. The sticky extracts obtained were placed at R.T. until it use.

2.2 Analysis of antioxidant activity index

Antioxidant capacity of the extracts was determined by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^.) method; antioxidant activity index (AAI) and IC₅₀ values were determined according to Sherer and Godoy.²¹

2.3 Sucrose-induced metabolic syndrome model

A total of 25 weaned male Wistar rats of 21 days age and 150–200 g body weight (Table 2) were individually housed and maintained in a 12-h light/dark cycle at 25 °C. This study was approved by the Research Committee of the Universidad Cristobal Colón following the guidelines of Mexican legislation, NOM-062-ZOO-1999²² for the care and use of laboratory animals. Animals were divided into two groups: the control group (CG; n = 5) was given a standard diet (Lab Diet 2004 S, Harlan Teklad Inc.) and water ad libitum; the Metabolic Syndrome group (MS; n = 20) which was given the same standard diet plus 40% sucrose in the drinking water ad libitum for 13 weeks to induce MS.

Table 2 Caloric intake and body weight of rats under the effect of P. karwinskii extracts after 13 days of experimental treatment.

Parameter	CG	MS	P	L	F
kcal/day (B)	65.10 ± 19.53	125.68 ± 22.62^a	87.43 ± 2.38	91.27 ± 10.38	81.30 ± 9.26
Initial weight (g)	372.0 ± 70.14	440.0 ± 83.19	321.67 ± 96.3	385.0 ± 69.28	431.67 ± 23.63
kcal/day (E)	79.05 ± 6.48	139.42 ± 6.77^a	128.35 ± 6.47^a	147.93 ± 31.13^a	147.78 ± 13.96^a
Final weight (g)	376.0 ± 77.73	464.50 ± 44.04	296.67 ± 88.8	365.0 ± 73.65	410.0 ± 20.00

CG: control group, MS: sucrose diet, P: sucrose diet and pseudobulb extract, L: sucrose diet and leaf extract, F: sucrose diet and flower extract, B: Beginning of the treatment, E: End of the treatment. The values represent the mean ± SD, superscript values show statistically significant difference as revealed by the Tukey’s test.

a P < 0.05.

2.4 Experimental diet and co-treatment

After MS induction this group (n = 20) was subdivided into 4 groups: MS group (n = 5) received a high caloric sucrose diet, P group (n = 5) received pseudobulb extract, L group (n = 5) received leaf extract, and F group (n = 5) received flower extract; these groups were compared with the control group (CG) as was mentioned above. P, L. and F groups received during 13 days by oral via, through a standard esophageal cannula, 200 mg/kg of body weight of the corresponding evaluated extract dissolved in water. Animals from all groups received the previously mentioned standard diet, during the same period of 13 days; additionally, for the MS group its diet included 40% sucrose in the drinking water ad libitum. At the end of the treatment the final weight was registered and a blood sample was taken from each rat with 18-h-fasted using cardiac puncture under anesthetic condition, prior to the killing of the animals. The blood was centrifuged and serum was kept at −20 °C until use.

2.5 Adipose tissue and Biochemical parameters

At the end of the treatment, rats were killed with anesthesia (0.1 ml intraperitoneal of 1% sodium barbiturate) to obtain the abdominal adipose tissue, epididymal and pericardial fat, and to determine the serum levels of glucose, cholesterol and triglyceride. These parameters were determined by enzymatic-colorimetric methods according to the manufacturer’s instruction using a biochromatic analyzer model Vitalab Selectra E.

2.6 Data analysis

Data obtained from the serum parameters, weight gain, organs and adipose tissue are presented as the mean ± SD. Data was evaluated using Minitab 16.1.0 software. Statistical significance was determined with an analysis of variance, a Tukey’s multiple range test was performed to test for significant differences between the different treatment groups. For all analyses the level of significance was P < 0.05.

3 Results

3.1 Total flavonoids and antioxidant activity of Prosthechea karwinskii extracts

Table 1 presents the concentration required to inhibit 50% of free radicals (IC₅₀), antioxidant activity index (AAI), and total flavonoid content of the extracts administered for biological evaluation.

Table 1 Antioxidant capacity of Prosthechea karwinskii extracts.

Extract	Yield w/w (%)	IC50	AAI
Pseudobulb	18.8 ± 1.335	43.06 ± 7.311	0.925 ± 0.162
Leaf	16.8 ± 2.335	6.91 ± 0.187	5.7 ± 0.157
Flower	33.6 ± 2.93	30.85 ± 2.51	1.276 ± 0.971

IC₅₀: 50% inhibitory concentration. AAI: antioxidant activity index = [DPPH]/IC₅₀.

The highest antioxidant capacity was showed by the extract of leaves (AAI = 5.7), followed by that from flowers (AAI = 1.276), and pseudobulbs (AAI = 0.925).

3.2 Caloric intake induced MS model

The daily caloric intake of the MS group compared with that of the control group (CG) during the 13 weeks MS induction period is shown in Fig. 1. The MS group showed significant differences from the CG due to sucrose consumption.

Fig. 1 Caloric intake during Metabolic Syndrome development. CG: control group, MS: sucrose diet, kcal: kilocalories. The values represent the mean ± DS ^∗P < 0.05.

Table 2 shows the caloric intake of CG, MS, P, L and F groups during 13 days of treatment consisting in the administration of the extracts and sucrose water. The initial weight for each evaluated group was nonuniform because evaluation of extracts began after induction of the animal model metabolic syndrome with 40% sucrose in drinking water; also, it must be considered the variation presented in the caloric intake of the animals during the development of MS, as is shown in Fig. 1.

Body weight of the CG increased by 1.1% while the body weight of the MS group increased by 5.6%. After the treatment, the P group lost the most weight, 7.8% of the total weight. Body weight of the L group decreased by 5.2% whereas that of the F group decreased by 5%, these results were respect to MS group.

3.3 Effect of Prosthechea karwinskii extracts in adipose tissue and serum parameters

Table 3 shows the effect of P. karwinskii extracts on abdominal, epididymal and pericardial adipose tissue weight over the 13 days experimental treatment.

Table 3 Abdominal, epididymal and pericardial adipose tissue weight and adiposity index after co-treatment with P. karwinskii extracts.

Parameter (g)	GC	MS	P	L	F
AT abdominal	12.20 ± 1.92	16.25 ± 5.91	9.33 ± 3.78^a	11.66 ± 2.30	11.0 ± 1.73^a
AT epididymal	10.0 ± 2.55	15.83 ± 6.67	6.66 ± 2.08^a	8.33 ± 2.88^a	10.33 ± 0.57^a
AT pericardial	0.62 ± 0.40	0.48 ± 0.18	0.70 ± 0.26	0.56 ± 0.30	0.40 ± 0.10
Total fat	22.82 ± 4.87	32.56 ± 12.76	16.69 ± 6.12^a	20.55 ± 5.48^a	21.73 ± 2.4^a

AT: adipose tissue, GC: control group, MS: sucrose diet, P: sucrose diet and pseudobulb extract, L: sucrose diet and leaf extract, F: sucrose diet and flower extract. The values represent the mean ± SD, superscript values show statistically significant difference as revealed by the Tukey’s test.

a P < 0.05.

In comparison with the MS group, the other groups experienced significant decreases in total adipose tissue: 49% in the P group, 36% in the L group, and 33% in the F group. According to the serum parameters of the CG and the MS group, glucose, cholesterol and triglycerides levels decreased in three groups: P, L and F (Table 4).

Table 4 Variation of serum parameters in Wistar rats after 13 days of treatment with P. karwinskii extracts.

Parameter (mg/dl)	CG	MS	P	L	F
Glucose	96.80 ± 10.64^a	135.26 ± 30.70	92.20 ± 7.95^a	64.12 ± 11.39^a	73.72 ± 25.76^a
Cholesterol	69.43 ± 10.39^a	87.52 ± 7.44	75.55 ± 0.32^a	76.04 ± 9.59^a	53.89 ± 10.54^a
Triglycerides	214.28 ± 18.07^a	259.28 ± 23.89	127.75 ± 46.56^a	108.78 ± 11.30^a	74.58 ± 22.35^a

CG: control group, MS: sucrose diet, P: sucrose diet and pseudobulb extract, L: sucrose diet and leaf extract, F: sucrose diet and flower extract. The values represent the mean ± SD, superscript values show statistically significant difference as revealed by the Tukey’s test.

a P < 0.05.

The leaf extract (L) had greatest effect in decrease the fasting glucose levels (64.12 mg/dl) in comparison with the MS group (135.26 mg/dl). The other extracts (F and P) also decreased glucose levels to 73.72 and 92.20 mg/dl, respectively. The flower extract (F) had the greatest effect on cholesterol levels (53.89 mg/dl) compared to the MS group (87.52 mg/dl); cholesterol levels in the CG were 69.43 mg/dl. P and L extracts also decreased this parameter but in a lesser extent: 75.55 mg/dl and 76.04 mg/dl, respectively. The extracts also had a reductive effect on triglyceride levels compared to the CG and MS groups, 214.28 and 259.28 mg/dl, respectively. The F group had the lowest triglycerides level (74.58 mg/dl), followed by L (108.78 mg/dl) and P (127.75 mg/dl) groups (Table 4).

4 Discussion

This work reveals by the first time that the pseudobulb, leaf and flower hydroalcoholic extracts from P. karwinskii ameliorated key parameters of the MS in a rat model and their relation with antioxidant capacity of the extracts. At the end of the treatment, the group given pseudobulb extract lost the greatest percentage of body weight, followed by the groups that received leaf and flower extracts. The results indicate that the evaluated extracts, administered in a dose of 200 mg/kg/day, significantly decreased adipose tissue mass and serum parameters levels that are associated with MS. The extracts evaluated here, not only reduced the adipose tissue mass but glucose levels as well; in contrast the results reported for aqueous extract from Ilex paraguariensis A. St.-Hil.²³ reduced abdominal and epididymal adipose tissue too, but raised glucose levels during 30 days of treatment. As the adiposity index data reveals, Prosthechea karwinskii extracts also had a greater effect on reducing adipose tissue than the previously reported for Salacia reticulate Wight²⁴ and Citrus grandis (L.) Osbeck.²⁵

The extracts also had a lowering effect on cholesterol and triglycerides levels; in this case, the flower extract had a greater effect than the reported for Camellia sinensis (L.) Kuntze.²⁶ Evaluation of Citrus grandis hydroalcoholic extract²⁵ indicates that after two weeks of treatment with a higher administered dose, cholesterol levels remained higher than our results. The dosage of leaf extract evaluated here had a greater glucose lowering effect than that reported for the polysaccharide obtained from Dendrobium chrysotoxum pseudobulbs⁸ using dosage of 200 mg/kg/day and 500 mg/kg/day. The leaf extract evaluated here also has a better dampening effect than the reported for Dendrobium denneanum polysaccharide⁹ using a dose of 300 mg/kg/day.

The antihyperglycemic effect of leaf extract in this work could be attributed to its greater antioxidant capacity compared with that for pseudobulb and flower extracts. Since hyperglycemia is associated with diminished endogenous antioxidants and increased oxidative stress, antioxidants have been shown to reduce the risk of hyperglycemia improving glucose disposal in the body.^5,27,28 According to the methodology described by Sherer and Godoy,²¹ these results show that P. karwinskii extracts have a higher AAI (Table 1) than other species reported in the following studies: ethanol-water leaves extract from Dendrobium speciosum Sm.²⁹ with IAA of 3.46 ∗ 10⁻⁵, the hydromethanolic and chloroform pseudobulb extracts from Prosthechea michuacana with an AAI of 0.0901 and 0.1268, respectively,¹³ the hydrometanolic rhizomes extract from Curculigo orchioides Gaertn.³⁰ with an AAI = 0.3720, the hydrometanolic leaf extract of Juglans regia L.³¹ with an AAI = 1.98 ∗ 10⁻⁹, and Jasminum humile L.³² with an AAI = 0.5598. Furthermore, this study confirms the potential antioxidant activity of compounds previously identified in P. karwinskii by Mijangos-Ricardez and López-Luna.¹⁷

Polyphenolic compounds also have been considered as a potential alternative for the treatment of MS given their effect in the absorption and metabolism of simple carbohydrates,^18,33 mainly reflected in the hypoglycemic and hypolipidemic effect of the extracts evaluated in this work. Recent studies have established that phenolic and flavonoid compounds are both capable of inhibiting lipid accumulation and apoptosis induction,³⁴ although they regulate the adipocyte physiology in a different way. An hypercaloric diet and genetic predisposition are the immediate causes of developing MS risk factors, such as obesity, insulin resistance, hyperlipidemia and hypertension, as well as of other metabolic diseases such as type II diabetes and cardiovascular disease.³⁵ In this work the hypercaloric diet (40% sucrose solution) administered at the beginning of the experiments was the principal factor who caused MS in the rats, thereby this work demonstrates that the extracts herein evaluated decreased the adiposity index and thus its relationship with associated metabolic disorders (hyperglycemia, hypercholesterolemia and hypertriglyceridemia).

Prosthechea karwisnkii extracts, by reducing adipose tissue, may also decrease characteristics MS serum parameters. This is because adipose tissue regulates the activation of macrophages, which favor the secretion of adipokines that regulates insulin resistance and the accumulation of triglycerides and cholesterol caused by increase of adiponectin and leptin secretions, in order to maintain equilibrium with the excess nutrients consumed by the organism.^34,35 For this reason, is require carry out studies that allow to evaluate the effect of the extracts on adipose tissue and dyslipidemia in a MS rat model, this research are actually in progress. In conclusion, the hydroalcoholic extracts studied hereof represent an alternative for the treatment of MS in the traditional medicine. Their reductive effect was showed even with the continued administration of a hyper-caloric diet to the groups treated with the extracts.

Conflict of interest statement

The authors declare that this article content has no conflicts of interest.

Acknowledgements

At the Consejo Nacional de Ciencia y Tecnología (CONACyT-Mexico), at Programa Nacional de Movilidad Estudiantil (ECOES) Santander for the scholarships awarded and Comisión de Operación y Fomento de Actividades Académicas (COFAA-IPN) for the support received. At the University Cristobal Colon for the facilities provided to use the vivarium, particular to M.V.Z. Christian Bautista Piña. Finally to the organizers of the Easter festivities in the Villa de Zaachila, Oaxaca and to the authorities of San Pedro and San Pablo Teposcolula, Oaxaca for the collection of plant material and the facilities provided.

Notes

Peer review under responsibility of Alexandria University Faculty of Medicine.

Available online 2 January 2017