Laboratorio de bioquímica: Escuela de Farmacia. Universidad de Puerto Rico, San Juan, Puerto Rico
Laboratorio de productos naturales: Escuela de Farmacia. Universidad de Puerto Rico, San Juan, Puerto Rico

Inhibition of aldose reductase by herbs extracts and natural substances and their role in prevention of cataracts

Dr. Ángel Guzmán¹ and Dr. Ricardo O. Guerrero²

Summary

Cataractogenesis is a common complication that occurs in diabetes mellitus. Aldose reductase is a lens enzyme probably involved in the development of this eye problem. The purpose of this investigation was to screen plant extracts for aldose reductase inhibitors (ARI) and to investigate their possible influence in diabetic cataractogenesis prevention. 13 plants and 3 natural products were randomly selected for our experiment.  The 19 extracts originated from plant material which was extracted with ethanol, water and DCM, and assessed for inhibitors of aldose reductase.  This enzyme was isolated from bovine lenses homogenates.  The enzyme was incubated in a reaction mixture containing 50 mM Na-phosphate buffer (pH 6.2), NADPH, 400 mM LiSO₄ and dl-glyceraldehyde. A spectrophotometrical assay was performed in which NADP is produced and its absorption read at 340 nm.  Eugenia borinquensis, Mangifera indica, Eucalyptus deglupta, and Syzygium malaccense were among the best inhibitors. Normal and diabetic Sprague Dawley rats were used to evaluate the in vivo effect of E. deglupta, M. indica, and E. borinquensis in cataractogenesis prevention. All of these extracts had preventive effects on the formation of cataracts. It is concluded that further investigation to explain the above findings is necessary. Perhaps the next step will include an activity monitored isolation of the effective principles. Future investigation will ince the isolation of the active principles in these extracts.

Key words: Puerto Rico, plant extracts, aldose reductase inhibition, cataractogenesis prevention.

A cataract is the opacity of the lens that produces painless gradual loss of vision. Acquired cataracts may result from trauma, radiation or metabolic disorders.  It may be produced in several conditions such as hyperglycemia, galactosemia, hypocalcaemia and diabetes mellitus.¹  Aldose reductase (AR), the key enzyme of the polyol pathway, is known to play important roles in diabetic complications.  The inhibitors of aldose reductase, therefore, would be potential agents in the prevention of one of these complications, namely, the development of cataracts.  Through the years, medicinal plants have been the focus of many investigations for the search of aldose reductase inhibitors (ARI). Ueda et al.² studying the leaves of Myrciaria dubia found ellagic acid and two of its derivatives as potent ARI.  Three flavonoids isolated from Brickellia arguta by Rosler et al.,³ showed anti-cataract activity in rats. Some Chinese herbs have also been investigated as source of ARI with a fluorometric assay.⁴ Kohda et al.⁵ discovered acteoside, an active ARI phenolic glycoside from a 70 % acetone extract of Monochasma savatierii.  Perillosides A and C were found to be excellent ARI.  These monoterpene glycosides were isolated from the leaves of Perilla frutescens.  The investigators⁶ attempted to establish a relationship between structure and inhibitory activity. Some sulfated flavonoids in Polygonum hydropiper were discovered to show potent inhibition against bovine lens aldose reductase.⁷ Other dihydroflavonols from Engelhardtia chrysolensis were determined to have potent aldose reductase inhibitory activity.^8,9 Other studies showed that flavonoid glycosides,¹⁰ isoflavonoids,¹¹and tannoid principles¹² had strong inhibitory activity.

The purpose of this investigation was to screen 13 plant extracts and 3 natural products for aldose reductase inhibitors (ARI) and to investigate their possible influence in diabetic cataractogenesis prevention.

Methods

Plant material

In the present study, the AR inhibitory activity of 19 extracts (see Table 1) was studied along with the in vivo effect of three of the best ARI for cataractogenesis prevention. The 19 extracts originated from 13 plant species and three natural products in different solvents. The natural products were acquired in local shops and the plant specimens were collected in the Caribbean National Forest (CNF) by Mr.  Luis Rivera (taxonomist of the CNF) and in the Botanical Garden of the University of Puerto Rico by Mr. Julio Figueroa (US Department of Agriculture) in 2003. Mr. Luis Rivera identified the plants and voucher specimens are maintained in the Herbarium of the School of Pharmacy. Extracts were made by macerating for several days 30-40 g of the ground plant material (leaves or natural product powder) with the solvent, filtering and evaporating the solvent in vacuo at 30-40⁰ C. Most of the extracts contained flavonoids, according to the magnesium and concentrated hydrochloric reaction.¹³ One of the plants, (E. borinquensis) is endemic to the cloud forests of the Luquillo mountains, Caribbean National Forest, north-eastern Puerto Rico.

Table 1. Herbs extracts and natural products evaluated for inhibition of aldose reductase

Plant	Scientific Name	Family	Solvent	Voucher
1	Allium cepa	Alliaceae o Amaryllidaceae	EtOH	PR007
2	Mangifera indica	Anacardiaceae	EtOH	PR001
3	Vaccinium myrtillus^1	Ericaceae	H2O	N/A
4	Vaccinium myrtillus^1	Ericaceae	EtOH	N/A
5	Nepeta cataria ^2	Lamiaceae	DCM	N/A
6	Nepeta cataria ^2	Lamiaceae	H20	N/A
7	Nepeta cataria ^2	Lamiaceae	EtOH	N/A
8	Clidemia octona	Melastomataceae	EtOH	PR153
9	Calycogonium squamulosum	Melastomataceae	EtOH	PR008
10	Psidium guajava	Myrtaceae	EtOH	PR046
11	Eugenia borinquenses	Myrtaceae	EtOH	PR060
12	Syzygium malaccense	Myrtaceae	EtOH	PR061
13	Eucalyptus deglupta	Myrtaceae	EtOH	PR059
14	Desmodium adscendens	Papilionaceae	EtOH	PR002
15	Coccoloba swartzii	Polygonaceae	EtOH	PR011
16	Manilkara bidentata	Sapotaceae	EtOH	PR029
17	Daucus carota	Umbelliferae	EtOH	PR114
18	Zingiber officinale	Zingiberaceae	EtOH	PR083
19	Curcuma longa^3	Zingiberaceae	EtOH	N/A

¹ Bilberry (Nature’s Herbs®) Modern Products, Inc. Milwaukee, WI 53209, USA,
² Catnip Herb (Nature’s Way Protects®) Springville, Utah 84663, USA,
³Turmeric (Spice Garden®) A Twin lab Division, American Fork, Utah 84003, USA,
 EtOH: Ethanol; DCM: Dichloromethane.

Bioassay

The bioassay procedure with some modifications was followed according to Haraguchi et al.⁷ Bioassay solutions of plant extracts were prepared by dissolving 2 mg of the extract in 50 µL DMSO. After 30 min or more, a 1 mL volume was completed by adding phosphate buffer pH 6.2. Aldose reductase was obtained from bovine eye lenses, kept frozen at -20^o C until the day of the experiment. The lenses were removed by lateral incision of the eye, washed with cold distilled water and kept cold. The lenses were deposited in a cold glass tube and the homogenized using a Teflon pestle in 3 volumes of cold 135 mM phosphate buffer (pH 7.0), containing 10 mM β-mercaptoethanol. The homogenate was centrifuged at 13,369 RPM (10 000 ´ g) for 15 min. The supernatant fluid was placed in a plastic 50 mL conical centrifuge tube and the protein in this fluid was then precipitated, by addition, drop by drop, of a solution 75 % (NH₄)₂SO_4. The pellet obtained by centrifugation was dissolved in the same buffer and used as enzyme preparation. This solution containing enzyme was analyzed using the Biuret reaction. Its protein concentration was 4 mg/mL.

The reaction mixture was prepared at 25^o C, with a total volume of 1.8 mL, containing 50 mM Na-phosphate buffer (pH 6.2), 0.125 mM NADPH, 400 mM LiSO₄, enzyme preparation equivalent to 4 mg protein and 3 mM dl-glyceraldehyde as a substrate with or without plant extract. The reaction was initiated by addition of NADPH and continued by 30 min. Absorbance readings were conducted at 340 nm absorption at the beginning and at the end of the reaction during this incubation time. NaHCO₃ 1M was added at the end of the 30 minute incubation period.  Quercetin (3.3 x 10^-1 to 33.3 mM), a known aldose reductase inhibitor was used as positive control to compare the plant extracts inhibitory activity. A negative control was prepared using 5 % DMSO in phosphate buffer (pH 6.2). The bioassays were run in triplicate and the average inhibitory activities are shown on table 2 as percentages. At the end, the inhibitory activity of the extracts was calculated using the following formula: 

                  

Results

Table 2 shows the percentage inhibition values of aldose reductase by the 19 extracts. Among the three evaluated natural products, C. longa, known as turmeric (75 %), and the EtOH extract of V. myrtillus known as bilberry (67 %), were found to have the best inhibitory activity.

The plant extracts of Eugenia borinquensis (82 %), Eucalyptus deglupta (88 %), and Mangifera indica (92 %) were among the best inhibitors of aldose reductase. Aqueous extracts of each of these plants (30 mg/mL) were assessed for prevention of cataractogenesis in streptozotocin induced diabetic rats (150-200 g) of the Sprague Dawley strain. Groups of ten rats were given plant extract to drink ad libitum for three months while controls received water. Groups were designated as A: rats with E. Borinquensis, B: rats with M. Indica, C: rats with E. deglupta, D: diabetic controls and E: non-diabetic controls. Three months later, significant effects were observed on the rats. It was visually established that all the rats treated with E. deglupta extract did not develop cataracts whereas 90 % of those tested with M. indica and 50 % of the animals treated with E. borinquensis did not develop cataracts, either.

          Table 2. Aldose Reductase Percentage Inhibition of herbs extracts and natural products

Plant	Scientific Name	Solvent	Aldose Reductase  Percent. Inhibition
1	Allium cepa	EtOH	1
2	Mangifera indica	EtOH	92
3	Vaccinium myrtillus	H2O	32
4	Vaccinium myrtillus	EtOH	67
5	Nepeta cataria	DCM	18
6	Nepeta cataria	H20	9
7	Nepeta cataria	EtOH	21
8	Clidemia octona	EtOH	51
9	Calycogonium squamulosum	EtOH	0
10	Psidium guajava	EtOH	40
11	Eugenia borinquensis	EtOH	82
12	Syzygium malaccense	EtOH	82
13	Eucalyptus deglupta	EtOH	88
14	Desmodium adscendens	EtOH	8
15	Coccoloba swartzii	EtOH	14
16	Manilkara bidentata	EtOH	0
17	Daucus carota	EtOH	0
18	Zingiber officinale	EtOH	14
19	Curcuma longa	EtOH	75

Discussion

The above results indicate presence of bioactive compounds in these three species that prevent cataractogenesis via a possible inhibition of aldose reductase. In relation to these three active plants we have found out that M. indica has been studied thoroughly. However, up to date only two investigations have been published in relation to diabetes mellitus. Mangiferin, a xanthone glycoside, isolated from the leaves of M. indica, was investigated and demonstrated to possess significant antidiabetic, antihyperlipidemic and antiatherogenic properties.¹⁴ In another study, it was found that the aqueous leaves extract of this plant possesses hypoglycemic activity.¹⁵ No information has been published on the aldose reductase inhibition of this plant. As for E. borinquensis, no information (chemical, pharmacological or ethnomedicinal) has been encountered. Nevertheless, it is interesting to note that an investigation on a member of this genus, Eugenia jambolana was found to prevent the development of cataracts in alloxan induced diabetic rats.¹⁶ In relation to E. deglupta, we have not been able to find, up to date, information concerned with anti-diabetic or anti-cataractogenic activities. It is concluded that further investigation to explain the above findings is necessary. Perhaps the next step will include an activity monitored isolation of the effective principles.

Acknowledgements

The authors are grateful to Mr. Luis Rivera of the Caribbean National Forest for his assistance in the identification of many specimens and for the collection of E. borinquensis. AG thanks San Juan Bautista School of Medicine and School of Pharamacy, University of Puerto Rico, for the economical support. ROG is indebted to “Proyecto Farmacia” for the assistance in the purchase of reagents and materials.

Resumen

Inhibición de la aldolasa reductasa por extractos de plantas y sustancias naturales y su papel en la prevención de las cataratas

La cataratogénesis es una complicación común que se produce en la diabetes mellitus. La enzima aldosa reductasa está relacionada probablemente con el desarrollo de esta enfermedad. El propósito de esta investigación consistió en determinar si extractos de plantas ejercen una actividad inhibitoria en la enzima y su posible efecto en la prevención de cataratas. Para estos fines, se seleccionaron al azar 13 plantas y 3 productos naturales. Los 19 extractos se originaron del material de las plantas extraídos con etanol, agua y diclorometano.  Los extractos fueron analizados por su inhibición de aldosa reductasa. La enzima se aisló de homogenizados de cristalinos de ganado vacuno. Este homogenizado se incubó luego en una mezcla de reacción que contenía amortiguador 50 mM  fosfato de sodio (pH 6,2), NADPH, 400 mM LiSO₄ y dl-gliceraldehido. Se realizó un análisis espectrofotométrico en el cual se producía NADP, se leyó su absorbancia a 340 nm, y se calculó el porcentaje de inhibición producido por los extractos. Los mejores inhibidores se encontraron en los extractos de Eugenia borinquensis, Mangifera indica, Eucalyptus deglupta y Syzygium malaccense. En otro experimento se usaron ratas Sprague Dawley normales y otras a las que se les indujo diabetes para estudiar el efecto in vivo de E. deglupta, M. indica y E. borinquensis en la prevención de la formación de cataratas. Todos estos extractos tuvieron efectos preventivos. Futuras investigaciones serán necesarias para   aislar los principios activos de estas plantas responsables de estos efectos.

Palabras clave: Puerto Rico, extractos de plantas, inhibición de aldosa reductasa, prevención de cataratogénesis.

References

1. Gittinger JW. Cecil Textbook of Medicine. 18th ed. Wyngaarden JB, Smith LH, editors. Philadelphia: WB Saunders & Co;1988.p.2289-90.