Laboratorio de productos naturales. Escuela de Farmacia. Universidad de Puerto Rico.

Specific bioassays with selected plants of Bangladesh

Dr. Ricardo O. Guerrero1, Dr. Mahmud T. H. Khan,2 Ms. Bárbara Casañas,3 y Ms. Mara Morales3

Summary

Bangladesh is an Asian country where only 20 per cent of the people are provided with modern healthcare services while the rest 80 per cent are dependent on traditional plant-based systems. Moreover, it is estimated that only 500 medicinal plant species had been recorded in Bangladesh out of 1,900 species regarded as having medicinal value. Purpose: Sixteen collections of medicinal plants of different families were extracted with several solvents (Ethanol 95%, water, chloroform, ethyl ether). The resulting extracts were subjected to five different specific bioassays: 1. Brine shrimp lethality test; 2. Antioxidant activity; 3. Inhibition of xanthine oxidase (XO); 4. Inhibition of ß-glucosidase, and 5. Inhibition of acetylcholinesterase. Methods. 1.The brine shrimp lethality test (BSLT) was conducted in 96-well microplates using serial dilutions. This examination uses the crustacean Artemia salina Leach and is predictive for cytotoxicity, as well as insecticidal, antifungal, and pesticidal activities. 2. A spectrometric method where the DPPH reagent (2,2-diphenyl-1-picrylhydrazyl), is mixed with serial dilutions of the extracts, was utilized to determine the antioxidant potential. 3. The xanthine oxidase inhibitory activity was measured using a spectrometric procedure. XO is the enzyme that acts as a catalytic agent in the conversion of xanthine to uric acid. Inhibition of this enzyme will decrease the blood levels of uric acid and result in antihyperuricemic effect. 4. The inhibition of ß-glucosidase was determined measuring spectrometrically the amount of liberated p-nitrophenol from the substrate p-nitrophenyl-ß-D-glucopyranoside. ß-glucosidase inhibitors such as castanospermine and 1-deoxynojirimycin interfere with the infectivity of HIV in vitro, as well as with syncytium formation. 5. The acetylcholinesterase inhibition activity was established with a spectrometric method. Inhibitors of this enzyme are valuable in the treatment of Alzheimer's disease and other disorders. Results: In the BSLT bioassay, Aphanamixis polystachya L. showed best activity with LD50 75.0 ppm, suggesting the presence of bioactive compounds in this extract. In the antioxidant bioassay, the same extract obtained an ED50 54.24 ppm. Other extracts that showed excellent free radical scavenging activity were Emblica officinalis Gaertn. (Syn. Phyllanthus emblica L.), ED50 132.16 ppm and Shorea robusta Gaertn., ED50 151.62 ppm. The XO inhibition produced several interesting results: best activity was shown by Shorea robusta Gaertn.: 60 % of inhibition, whereas Emblica officinalis Gaertn. displayed 48 % inhibition. Inhibition of the ß-glucosidase enzyme generated acceptable results: 63 % and 45 % with the extracts of S. robusta and E. officinalis, respectively. Finally, in the acetylcholinesterase inhibition, none of the plant extracts showed positive results. Conclusions. As demonstrated by the data, there are some extracts that presented several biological activities. It will be necessary to pursue further investigation to bring about the identification of the responsible metabolites for these activities.

Key Words: Bangladesh, plant extracts, brine shrimp lethality test, antioxidant activity, xanthine oxidase inhibition, ß-glucosidase inhibition, acetylcholinesterase inhibition.

According to the World Health Organization, almost 80 % of the world's inhabitants use traditional medicine in their health primary care. This scenario is similar to the one occurring in Bangladesh. Bangladesh is an Asian country where only 20 % of the people can be provided with modern healthcare services while the rest 80 % are dependent on traditional plant-based systems. The use of traditional medicine is increasing in developing countries. This is probably due to the escalating in population, to the government support to the forms of indigenous medicine, and finally, to the patriotic desire to revive and maintain the traditional culture. There are several studies on the botanical aspects of the plants of Bangladesh. However, although plants are used by a great segment of the population, scarce investigation has been done on their biological activities. Moreover, it is estimated that only 500 medicinal plant species had been recorded in Bangladesh out of approximately 1900 species regarded as having medicinal value.1

METHODS

Plant material

For our study, 16 species of medicinal plants (14 families) were collected in different places of Bangladesh. All of these plants have a history of medicinal use. Different plant parts (whole plant, stem bark, seeds, fruit, root, nuts) were collected and extracted with solvents (ethanol 95%, water, chloroform, ethyl ether). See Table 1. Plants were identified by Dr. J. Uddin (BCSIR Lab.) and Dr. M.S.K. Choudhuri (Jahangirnagar University). Voucher specimens were stored in the herbarium of the Faculty of Pharmaceutical Sciences, University of Science and Technology, Chittagong, Bangladesh. Approximately, 200 g of dry weight plant material was macerated at room temperature for 24 h with the solvent and filtered. The solvent was removed by rotary evaporation under reduced pressure and the resulting extracts were submitted to the laboratory of Natural Products, School of Pharmacy, University of Puerto Rico. The extracts were subjected to five in vitro bioassays.

Bioassays

Five biological activities were studied in vitro with the extracts of these plants: 1. Brine shrimp lethal toxicity (BSLT); 2. Antioxidant capacity; 3. Inhibition of xanthine-oxidase (XO); 4. Inhibition of ß-glucosidase, and 5. Inhibition of acetylcholinesterase. All the assays were performed in triplicate.

1. Brine Shrimp Lethal Toxicity (BSLT)

BSLT is a primary toxicity screening procedure used as an initial screening of bioactive compounds. Since 1956 this bioassay has been utilized for environmental studies, testing of natural toxins, and as a general screening for bioactive substances in plant and marine extracts. The protocol by Solís et al.,2 using serial dilutions in 96-well micro-plates was followed throughout our experiments. Berberine HCl (Sigma) served as positive control. The probit analysis method described by Finney3 was used to calculate LC50 values and 95 % confidence intervals.

2. Antioxidant activity

Free radicals are associated to health problems such as cancer, aging, and heart disease. Free radical scavenging activity was evaluated with the method of Joyeux et al.4 A spectrometric method where the DPPH reagent (2,2-diphenyl-1-picrylhydrazyl), is mixed with serial dilutions of the extracts, was utilized to determine the antioxidant potential. Quercetine was used as positive control. DPPH and quercetine were purchased from Sigma. Again, probit analysis served to calculate ED50 values and 95 % confidence intervals.

3. Inhibition of xanthine-oxidase (XO)

The xanthine oxidase inhibitory activity was measured using a spectrometric procedure according to Noro et al.5 XO is the enzyme that acts as a catalytic agent in the conversion of xanthine to uric acid. Inhibition of this enzyme will decrease the blood levels of uric acid and result in antihyperuricemic effect. Uric acid is normally dissolved in the blood. When the concentration rises, uric acid forms crystals in the joint. The crystals set up the inflammation process called acute gout. The enzyme (XO), the substrate (xanthine), and the inhibitor (allopurinol) were purchased from Sigma. Allopurinol was used as a positive control. The obtained results show the percentage of enzyme inhibition.

4. Inhibition of ß-Glucosidase

The inhibition of ß-glucosidase was determined measuring espectrometrically the amount of liberated p-nitrophenol from the substrate p-nitrophenyl-ß-D-glucopyranoside. It is known that castanospermine and 1-deoxynojirimycin hydrochloride block HIV-induced syncytium formation and also interfere with HIV infectivity in vitro. Castanospermine (1,6,7,8 tetrahydroxyoctahydroindolizine) is an alkaloid isolated from the seeds of the tree Castanospermun australe and is known to be a potent ß-glucosidase inhibitor.6 It seems reasonable therefore to use the ß-glucosidase inhibition procedure to identify castanospermine or 1-deoxynojirimycin-like activities. The procedure of Saul et al.,7 was followed in our work. 1-deoxynojirimycin HCl (Sigma) was used as positive control. A negative control was utilized to eliminate the interference of the color of the extract. The results were expressed as the percentage inhibition of the enzyme. The substrate (p-nitrophenyl-ß-glucopyranoside) and the enzyme, ß-glucosidase were acquired from Sigma.

5. Inhibition of acetylcholinesterase

A spectrometric assay was utilized to measure the acetylcholinesterase inhibition activity.8 This enzyme is in charge of the hydrolysis of the cationic neurotransmitter acetylcholine, in the central and peripheral nervous system. Due to the key function of acetylcholinesterase in the nervous system, it has been an appealing target for the discovery of inhibitors. Some of them are being used in the treatment of Alzheimer's disease, ataxia, senile dementia, and for enhancing the long term memory processes. The substrate (acetylthiocholine), the enzyme (acetylcholinesterase) and the color precursor reagent (dithiobisnitrobenzoic acid) were purchased from Sigma. Donepezil hydrochloride (Aricept® tablet, Eisai Inc. and Pfizer Inc.) served as positive control. The results of the enzyme activity were calculated as the percentage inhibition.

RESULTS

The sixteen medicinal plants evaluated in this screening are shown in table 1. They are distributed among 14 genera and 14 families. Table 2 summarizes the results of the bioassays.

Table 1. Bangladesh plant extracts

Scientific Name	Family	Common name	Plant part	Voucher	Extract
Lagerstroemia speciosa	Lythraceae	Jarul	Stem Bark	BCS-343	CHCl3
Moringa oleifera	Moringaceae	Sajna	Stem Bark	BCS-4575	Ethanol
Hygrophila auriculata	Acanthaceae	Talmakna	Seeds	AK-4233	Ethanol
Paederia foetida	Rubiaceae	Gondhovaduley	Whole plant	25372	Ethanol
Aegle marmelos	Rutaceae	Bel	Stem Bark	BCS-243	Ethanol
Shorea robusta	Dipterocarpaceae	Shal, Gajari	Stem Bark	BC-5498	H20
Emblica officinalis	Euphorbiaceae	Amalaki	Dried fruits	BC-3823	Ethanol
Tribulus terrestris	Zygophylaceae	Gokkhur	Dried seeds	EPL-032	Ethanol
Vernonia anthelmintica	Asteraceae	Somraji	Dried seeds	DGF-8913	Ethanol
Nigella sativa	Ranunculaceae	Kalajira	Dried seeds	SDK-84	Ethanol
Aphanamixis polystachya	Meliaceae	Royna	Dried stem bark	GDK-8913	Ethanol
Polyalthia longifolia	Annonaceae	Debdaru	Root	GFS-738	Ethanol
Semecarpus anacardium	Anacardiaceae	Bhela	Dried nuts	EPL-012	Ethanol
Nigella sativa	Ranunculaceae	Kalajira	Dried seeds	SDK-84	Diethyl ether
Hemidesmus indicus	Asclepiadaceae	Anatamul, Sarsaparilla, Shariba	Root	BC-1083	Ethanol
Aegle marmelos	Rutaceae	Bhela	Dried nuts	BCS-243	Ethanol

Table 2.Biological activities results

Scientific Name	BSLT(LC50: ppm)	Antioxidant activity (ED50: ppm)	XO inhibition(Percentage)	ß-Glucosidase inhibition (Percentage)	Acetyl-cholinesterase inhibition (Percentage)
L. speciosa	>1,000	>1,000	**	0	**
M. oleifera	>1,000	>1,000	4.90	1.41	0
H. auriculata	126.5(145.0-109.1)*	>1,000	11.52	11.33	0
P. foetida	>1,000	>1,000	0.80	15.47	0
A. marmelos (Stem bark EtOH extract)	>1,000	>1,000	10.80	23.56	0
S. robusta	>1,000	151.62(300.1-98.2)*	59.61	62.65	0
E. officinalis	>1,000	132.16(256.4-84.6)*	47.77	45.00	0
T. terrestris	>1,000	>1,000	0	7.68	0.3597
V. anthelmintica	>1,000	>1,000	0.57	0	0.0711
N. sativa (EtOH extract)	>1,000	>1,000	11.26	0	0.0166
A. polystachya	75.00(84.7-65.4)*	54.24(93.0-35.6)*	7.04	13.67	0
P. longifolia	>1,000	>1,000	0.80	5.20	0
S. anacardium	>1,000	37.24(50.7-28.1)*	0	3.98	0.1247
N. sativa (Et2O extract)	>1,000	>1,000	7.12	0	0
H. indicus	>1,000	>1,000	0.38	0	0.7823
A. marmelos (Dried nuts EtOH extract)	>1,000	>1,000	**	4.38	0

* Lower and Upper Confidence Limits    ** Not enough extract

DISCUSSION

From the 16 extracts evaluated in the BSLT, only 2 (12.5%) displayed LC50 >150 ppm. The seeds of Hygrophylla auriculata presented LC50 126.5 ppm whereas the stem bark of Aphanimix polystachya showed LC50 75 ppm. These results suggest the presence of bioactive plant metabolites. H. auriculata is used in the Indian system of medicine for the treatment of liver ailments and a significant hepatoprotective activity of the seeds has been reported.9 Similarly, a crude ethanolic extract of A. polystachya has shown beneficial effects on toxic liver injury.10

It is interesting to notice that the extract of A. polystachia also exhibited free radical scavenging activity in the antioxidant assay (ED50: 54.2 ppm). Other extracts that showed excellent activity in this evaluation were Emblica officinalis Gaertn. (Syn. Phyllanthus emblica L.) ED50: 132.1 ppm, Shorea robusta Gaertn. ED50: 151.62 ppm. Semecarpus anacardium presented the best activity, ED50: 37.2 ppm. Some interesting studies have been carried out with this plant. The alcoholic extract of the nuts of this plant has shown dose dependent antifungal activity in vitro against Aspergillus fumigatus and Candida albicans.11 Also, the ethanolic extract of the fruits of S. anacardium has demonstrated a significant reduction of sperm motility and density in albino rats.12

The xanthine oxidase testing produced a 60 % inhibition with Shorea robusta extract and 48 % inhibition with Emblica officinalis. The bioassay on the inhibition of ß-glucosidase presented relatively good results. The same plant extracts, S. robusta and E. officinalis showed 62.6 % and 45.0 % inhibition, respectively. These results are considered interesting for future examination of these plants. In Bangladesh, the stem bark of S. robusta is used for its antibacterial and antiviral properties. It is purported to have stimulant and aphrodisiac activities.13 E. officinalis has been the target of many studies. Among them, it has been found that its extract displayed antitumor activity on human breast tumor cell lines.14 In another study, the chronic oral administration of the fruit protected rat hearts from oxidative stress,15 and an antitussive effect was demonstrated in cats.16 Other experiment performed in mice, suggested that this plant ameliorates hyperthyroidism with an additional hepatoprotective effect.17 Antiinflamatory18 and antihypercholesteremic19 activities in rats have also been reported.

None of the plant extracts inhibited acetylcholinesterase in a categorical manner. The values of inhibition did not even reach 1 %.

Overall, extracts from 2 plants (12.5 %) showed activity in 3 of the 5 bioassays. S. robusta and E. officinalis had mild antioxidant activity (ED50s 151.62 and 132.16 ppm), and good activity in the inhibition of xanthine oxidase (59.61 % and 47.77 %), as well as in the inhibition of ß-glucosidase (62.65 % and 45.00 %). These facts make these plants worth as potential sources of bioactive compounds. A. polystachia extract displayed activity in two bioassays: in the BSLT, LC50 of 75.00 ppm; and in the free radical scavenging experiment, ED50 54.24 ppm. These figures indicate that this plant would be a possible candidate for further research to examine the active metabolites. Finally, S. anacardium and H. auriculata presented bioactivity in one of the five bioassays. S. anacardium had the best antioxidant activity (ED50 37.24 ppm), and H. auriculata manifested some activity in the brine shrimp toxicity experiment (LC50 126.5 ppm). All of these results imply the existence of bioactive metabolites and therefore, more research with all of these plants is warranted. All the active plants are used in traditional medicine in the Indian region.13 0

Acknowledgements

ROG thanks "Proyecto Farmacia" for the economic support of this study. The invaluable help of students of Pharmacy Iliana E. Sánchez and Marlén Flores in this investigation is appreciated.

Resumen

Antecedentes: Bangladesh es un país de Asia en donde sólo al 20 % de su población se le provee con servicios modernos de cuidado de la salud, mientras que el 80 % restante depende de sistemas tradicionales basados en plantas medicinales. Además, se estima que solamente 500 plantas medicinales han sido estudiadas de un total de 1 900 que se cree tienen valor medicinal. Propósito: 16 especies de plantas medicinales pertenecientes a diversas familias se extrajeron con diferentes solventes (etanol 95 %, agua, cloroformo, éter etílico). Los extractos se sometieron a 5 bioensayos específicos diferentes: 1. Examen de mortalidad de los camarones salinos, 2. Actividad antioxidante, 3. Inhibición de la xantina oxidasa, 4. Inhibición de la ß-glucosidasa e 5. Inhibición de la acetilcolinesterasa.
Métodos: 1. El examen de mortalidad de los camarones salinos fue realizado en microplatos de 96 pozos con diluciones seriadas. Este bioensayo usa el crustáceo Artemia salina Leach y sus resultados correlacionan con la citotoxicidad, así como también con actividades insecticidas, antifúngicas y pesticidas. 2. La actividad antioxidante se determinó con un método espectrofotométrico en donde diluciones seriadas de los extractos se mezclaron con el reactivo 2,2-diphenyl-1-picrilhydrazilo. 3. La actividad inhibitoria de la xantina oxidasa se midió usando un procedimiento espectrofotométrico. Xantina oxidasa es la enzima que actúa como agente catalítico en la conversión de xantina a ácido úrico. La inhibición de esta enzima disminuye los niveles sanguíneos de ácido úrico resultando en un efecto antihiperuricémico. 4. La inhibición de ß-glucosidasa se estableció midiendo espectrofotométricamente la cantidad de p-nitrofenol liberado del sustrato p-nitrofenil-ß-D-glucopiranósido. Inhibidores de ß-glucosidasa tales como castanospermina y 1-desoxinojirimicina intefieren con la infectividad de HIV in vitro, así como también con la formación del sincitio. 5. La actividad de inhibición de acetilcolinesterasa fue establecida con un método espectrométrico. Los inhibidores de esta enzima son valiosos en el tratamiento de la enfermedad de Alzheimer y otros desórdenes.
Resultados: En el bioensayo de mortalidad de los camarones salinos , Aphanamixis polystachya L. exhibió la mejor actividad con un LD50 75,0 ppm, sugiriendo la presencia de compuestos bioactivos en el extracto. En el procedimiento antioxidante, el mismo extracto obtuvo un ED50 54,24 ppm. Otros extractos mostraron excelente actividad de eliminación de radicales libres: Emblica officinalis Gaertn. (Syn. Phyllanthus emblica L.), ED50 132,16 ppm y Shorea robusta Gaertn., ED50 151,62 ppm. La inhibición de xantina oxidasa produjo algunos resultados interesantes: Shorea robusta Gaertn. demostró la mejor actividad: 60 % de inhibición, mientras que Emblica officinalis Gaertn. exhibió 48 % inhibición. La inhibición de la enzima ß-glucosidasa generó resultados aceptables: 63 y 45 % con los extractos de S. robusta y E. officinalis, respectivamente. Finalmente, en la inhibición de acetilcolinesterasa, ninguno de los extractos de plantas presentó resultados positivos.
Conclusiones. De acuerdo a los resultados, algunos de los extractos mostraron actividades biológicas. Sería importante continuar con la investigación para identificar a los metabolitos responsables de estas actividades.

Palabras clave: Bangladesh, examen de mortalidad de los camarones salinos, actividad antioxidante, inhibicion de xantina oxidasa, inhibicion de ß-glucosidasa, inhibicion de acetilcolinesterasa.

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Recibido:29 de abril de 2004. Aprobado: 12 de mayo de 2004.
Dr. Ricardo O. Guerrero. School of Pharmacy, Medical Sciences Campus, University of Puerto Rico, P.O. Box 5067, San Juan, PR 00936-5067.

1Ph.D. in Pharmacognosy.
2 Ph.D. in Pharmacology.
3 Fourth year Chemistry student.