Biological Evaluation of Some Selected Plant Species of Pakistan

Abstract

Seven methanol extracts from five different plant species [Salvia nubicola. B. (Laminiaceae), Acer oblongifolium. D. (Aceraceae), Sorbaria tomentosa. L. (Rosaceae), Hedera nepalensis. K. (Araliaceae), and Artemisia fragrans. W. (Asteraceae)] were evaluated for brine shrimp cytotoxicity, antitumor potato disc, and radish seed phytotoxicity activity. Four of the seven extracts revealed significant ED₅₀ value ranging from 11.9 to 226.8 ppm. Inhibition of tumor formation ranged from 9 to 82.9% by all extracts in antitumor potato disc assay at three different concentrations tested (1000, 100, and 10 ppm). Growth inhibition was observed by all extracts in radish seed bioassay at high concentration (10,000 ppm). At low concentration (1000 ppm), three extracts from two plant species (leaf and flower extract of S. nubicola., stem extract of S. nubicola., and stem extract of H. nepalensis.) presented stimulation of growth ranging from 3.5 to 43.2%. A positive correlation was observed in the results of three of the described assays.

Keywords:

• Antitumor
• bioassays
• chemotherapeutic
• cytotoxic
• phytotoxicity

Introduction

For this study, medicinally important plant species of Northern areas of Pakistan were selected on the basis of traditional knowledge, i.e., they are used by local healers for the treatment of various diseases.

Salvia nubicola. B. belongs to the family Laminiaceae. A number of Salvia. species are used in folk medicine for the treatment of dysentery, boils, fall injuries, hepatic problems, and cancer (Fujita & Node, 1984; Zhang & Li, 1994). Acer oblongifolium. L. (Aceraceae) is also a plant species evaluated in the present study. Several species of Acer. genera are used traditionally in the treatment of cancer, polio, and dysentery (Moerman, 1998). Hamayun et al. (2005) described Sorbaria tomentosa. L. (Rosaceae) as a medicinal plant. Hedera nepalensis. L. (Araliaceae) is considered an antidiabetic in folk medicine (Gilani et al., 2001). Ahmed et al. (2004) described the ethnobotanical importance of Artemisia fragrans. W. (Asteraceae). According to their report, leaf extract of Artemisia fragrans. has anthelmintic activity and is also used against wounds, earache, toothache, and asthma.

During the study of medicinal plants, prior to fractionation and structural elucidation of individual components of botanical extracts, it is necessary to evaluate their biological activity. Several bench top assays, such as brine shrimp cytotoxicity assay, antitumor potato disc assay, and radish seed phytotoxicity assays, can be used as major prescreening assays in this regard.

The brine shrimp cytotoxicity assay is a rapid, inexpensive assay requiring no special technical training. A positive correlation between brine shrimp toxicity and KB (human nasopharyngeal carcinoma) has been found (McLaughlin & Rogers, 1998). Moreover, this assay has been used successfully to biomonitor the isolation of cytotoxic (Siqueira et al., 2001), antineoplastic (Badaway & Kappe, 1997), antimalarial (Perez et al., 1997), insecticidal (Oberlies et al., 1998), and anti-feedant (Labbe et al., 1993) compounds from plant extracts.

Crown gall is a neoplastic disease of plants induced by specific strains of the Gram-negative bacterium, Agrobacterium tumefaciens.. Galsky et al. (1980) demonstrated that inhibition of crown gall initiation on potato discs showed apparent agreement with compounds and plant extracts known to be active in 3PS (in vivo., mouse leukemia) antitumor assay. Coker et al. (2002) reported the inhibition of tumor induction by means of different antineoplastic drugs. They further demonstrated that A. tumefaciens.-induced potato disc assay was an effective indicator of antitumor activity regardless of the mechanism of drug action.

Evaluation of herbicidal or growth stimulation properties of plant extracts requires another simple benchtop assay. Radish seed bioassay has been used previously for phytotoxic evaluation of plant extracts (Turker & Camper, 2002). Radish seeds are easily available, and the assay does not require special technical training.

Brine shrimp lethality assay and phytotoxic evaluation of botanical extracts present predictions for various types of biological activities. Potent tumor inhibition in antitumor potato disc assay by crude botanical extracts confirms this potential and can open new avenues toward the discovery of anticancer drugs. Growth stimulation or inhibition in radish seed phytotoxicity assay can determine the herbicidal or growth stimulatory potential of the plant extracts tested. A positive correlation between these assays can determine pharmacological importance of medicinal plants.

Major objectives of the present study involve evaluation of biological activities of crude plant extracts and determination of any possible correlation between three types of biological assays.

Materials and Methods

Plant species were collected from northern areas of Pakistan (Swat and Kalam districts). Identification was carried out in the taxonomy laboratory of the Department of Plant Sciences, Quaid-i-Azam University, Islamabad, and voucher specimens were deposited here.

Preparation of methanol extracts

Methanol extracts were prepared by maceration procedure. Plant material was separated into different parts (leaves, stems, and roots), dried under shade, and, finally, powdered. Powdered material was soaked in methanol for 7 days, then filtered and evaporated in a rotary evaporator (Buchi Rotavapor R-200, Switzerland). Extracts were stored at − 20°C. Various extracts used in the present study are described in Table 1.

Table 1.. List of plant species with respective plant extracts.

Plant species	Family name	Methanol extracts
Salvia nubicola.	Laminiaceae	(L + F) S. nubicola. (methanol extract of leaves and flowers of S. nubicola.)
		(S) S.nubicola. (methanol extract of stem of S. nubicola.)
Acer oblongifolium.	Aceraceae	(L + S) A. oblongifolium. (methanol extract of leaves and stem of A. oblongifolium.)
Sorbaria tomentosa.	Rosaceae	(L + S) S. tomentosa. (methanol extract of leaves and stem of S. tomentosa.)
Hedera nepalensis.	Araliaceae	(L + S) H. nepalensis. (methanol extract of leaves and stem of H. nepalensis.)
Artemisia fragrans.	Asteraceae	(L + F) A. fragrans. (methanol extract of leaves and flower of A. fragrans.)
		(R) A. fragrans. (methanol extract of roots of A. fragrans.)

Brine shrimp cytotoxicity assay

Brine shrimp cytotoxicity assay was performed according to the standard procedure described by McLaughlin (1991). Three concentrations (1000, 100, and 10 ppm) of plant extracts were used in this assay. Brine shrimp larvae were hatched in a small partitioned tank in artificial seawater. Illumination was provided on one side to attract newly hatched larvae. Brine shrimp larvae with second instar stage were used in this assay.

Plant extracts of respective concentrations were added to dram vials. To each dram vial, 10 brine shrimp larvae were added. Negative control was prepared by evaporating 0.5 ml of methanol in dram vials and then adding sea salt solution to it. Following 24 h of incubation, survivors were counted using a magnifying glass. The experiment was repeated three times. Mortality data was transformed by Probit analysis in a finny computer program to estimate ED₅₀ value. Percentage of mortality was also calculated at all concentrations.

Antitumor potato disc assay

Antitumor potato disc assay was performed according to standard procedure described by McLaughlin and Rogers (1998). A 48 h bacterial culture of At 10 strain of Agrobacterium tumefaciens. was used in this experiment. Inoculum with three concentrations of test samples (1000, 100, and 10 ppm) was prepared containing bacterial culture and autoclaved distilled water.

Red-skinned potatoes were purchased from a local market and surface sterilized by using 10% bleach solution. A borer of 8-mm diameter was used to bore out potato cylinders. Cylinders were cut into 2-mm discs. Autoclaved agar solution (1.5%) was poured into Petriplates and solidified. Ten discs were placed on the agar surface of each plate, and 50 µl of inoculum was placed on the surface of each disc. The plates were sealed with parafilm to avoid contamination and moisture loss. The plates were incubated at 28°C in an incubator in the dark. The experiment was carried out in strict sterilized conditions and repeated in triplicate. After 21 days of incubation, potato discs were stained with Lugol's solution (10% KI, 5% I₂) and tumors were counted under a dissecting microscope with side illumination. Tumor inhibition was calculated using following formula: where ns = number of tumors in sample, nc = number of tumors in control.

More than 20% tumor inhibition is considered significant (Ferrigini et al., 1982). Data was statistically analyzed using ANOVA.

Antibacterial assay against Agrobacterium tumefaciens.

Antibacterial assay was performed according to standard agar well-diffusion method. A 24 h bacterial culture of At 10 strain of A. tumefaciens. was used in this experiment. Bacterial culture was mixed with autoclaved nutrient agar medium, poured in a Petri plate, and solidified. Wells were prepared by using an 8-mm borer and sealed with nutrient agar medium. A volume of 100 µl of the plant extract at 1000 ppm concentration was used. Roxythromycin (1000 ppm) and cefixime (1000 ppm) were used as positive control simultaneously. The plates were incubated at 28°C for 48 h, and results were recorded. Minimum Inhibitory Concentration (MIC) was determined by using serial dilution method (Ansari et al., 2005).

Radish seed phytotoxicity assay

The experiment was conducted according to standard procedure described by Turker and Camper (2002). It consisted of two parts. In part one, two concentrations (10,000 and 1000 ppm) of the plant extracts were prepared in methanol. Filter papers (Whatman #1) were placed in Petri plates, and 5 ml of each concentration was added. Methanol was evaporated, and 5 ml of distilled water was added. Twenty radish seeds surface sterilized with 0.1% mercuric chloride were placed in each Petri plate. The plates were sealed with parafilm to avoid moisture loss and incubated at 23 ± 2°C. In control plates, 5 ml of methanol was added and evaporated. Root length was measured on the third and fifth day of incubation. The experiment was repeated in triplicate.

In the second part of the experiment, two concentrations of the plant extracts (7500 and 1000 ppm) were used. The procedure for the second part is similar to that of the first part except for the concentrations of extracts and number of seeds. In the second part of the experiment, 100 radish seeds were added to each plate. Germinated seeds were counted every day from the first to the fifth day. The experiment was repeated in duplicate. Both experiments were carried out in strict sterilized conditions. Results were statistically analyzed by using ANOVA. Statistical analysis of radish seed bioassay data was performed only on fifth-day data.

Results

Brine shrimp cytotoxicity assay

Four of the seven extracts tested exhibited ED₅₀ less than 1000 ppm in brine shrimp assay, indicating potent cytotoxic activity of these extracts. ED₅₀ in these extracts ranged from 11.9 to 226.8 ppm (Table 2).

Table 2.. Illustration of percentage mortality of brine shrimp at different concentrations of extracts and respective ED₅₀ values.

Methanol extracts	1000 ppm (%)	100 ppm (%)	10 ppm (%)	ED50 (ppm)
(L + F) S. nubicola.	13.3	10	10	> 1000
(S) S. nubicola.	10	6.6	6.6	> 1000
(S) A. oblongifolium.	66.70	16.70	6.70	226.8
(L + S) S. tomentosa.	13.40	14	10	> 1000
(L + S) H. nepalensis.	100	56.6	23.3	47.7
(L + F) A. fragrans.	100	66.6	46.6	11.99
(R) A. fragrans.	100	60	46.6	19.7

Results for percentage mortality of brine shrimp indicate that the highest percentage mortality was observed at 1000 ppm by most of the extracts tested. At 100 ppm, only three extracts (L + F) A. fragrans., (R) A. fragrans., and (L + S) H. nepalensis., presented a significant mortality rate, i.e., 66.6, 60, and 56.6% respectively.

Antitumor potato disc assay

All extracts exhibited tumor inhibition at the three concentrations tested. Tumor inhibition was observed in a concentration-dependent mode. Statistical analysis using ANOVA showed that the effect of concentration and extract was highly significant. The effect of interaction of concentration and extract factors is presented in Table 3 with the respective rank order obtained. Extract of leaves and stem of A. oblongifolium. presented the highest percentage of tumor inhibition at all concentrations.

Table 3.. Average number of tumors produced at different concentrations of extracts. Values with similar letters are not significantly different from each other at p > 0.05.

Extracts	Concentrations (ppm)	Average number of tumors per disc	Percentage of tumor inhibition
(L + F) S. nubicola.	1000	3.4 ± 0.8^i.	61.3
	100	6 ± 0.9^e.	31.8
	10	8 ± 0.2^b.	9
(S) S. nubicola.	1000	3.3 ± 0.4^i.	62.5
	100	5.1 ± 0.7^g.	42.5
	10	5.7 ± 0.5^f.	35.2
(L + S) A. oblongifolium.	1000	1.5 ± 0.5^k.	82.9
	100	3.4 ± 0.6^i.	61.3
	10	3.6 ± 0.8^i.	59
(L + S) S. tomentosa.	1000	2.3 ± 0.5^j.	73.8
	100	3.7 ± 0.6^i.	57.9
	10	4.8 ± 0.4^h.	45.5
(L + S) H. nepalensis.	1000	5.3 ± 1.07^fg.	39.1
	100	6.7 ± 0.8^d.	22.9
	10	7.1 ± 1.1^c.	20.2
(L + F) A. fragrans.	1000	4.7 ± 0.3^h.	46.4
	100	5.4 ± 0.2^f.	38.5
	10	6.9 ± 0.3^c.	20.4
(R) A. fragrans.	1000	5.7 ± 0.3^f.	34.6
	100	6.6 ± 0.4^d.	24.4
	10	7.4 ± 0.3^c.	15.7
Control		8.8 ± 0.9^a.

Superscript letters ranging from a. to k. indicate respective Least Significant Difference rank orders of mean values.

Antibacterial assay against Agrobacterium tumefaciens.

The effect of extracts on viability of A. tumefaciens. was evaluated by using the agar well-diffusion method. None of the six extracts tested showed any significant effect on viability of A. tumefaciens.. One of the seven extracts, i.e., (L + S) S. tomentosa., was slightly effective against A. tumefaciens. at 1000 ppm (zone size = 10 mm, MIC = 0.8 mg/ml).

Radish seed phytotoxicity assay

The effect of two different concentrations (10,000 and 1000 ppm) of the extracts was studied on root growth inhibition or stimulation of radish seedling. All extracts inhibited root growth at 10,000 ppm. The highest percentage of inhibition was observed by leaf and stem extract of Sorbaria tomentosa.. In three of the extracts, root growth stimulation was observed at 1000 ppm (Fig. 1). Leaf and stem extract of Hedera nepalensis. presented the highest stimulation of root length at 1000 ppm.

Figure 1 Effect of two different concentrations [(a) 10,000 ppm and (b) 1000 ppm] on root length. Values with similar letters do not show significant difference; p > 0.05.

In a second experiment, the effect of two different concentrations of each extract (7500 and 1000 ppm) on seed germination was observed as a function of incubation period of seeds. A gradual increase in seed germination for all extracts was observed until the secnod day of incubation. The effect of concentrations remained significant, and inhibition of seed germination was observed in the case of all extracts at 7500 ppm (Fig. 2). Leaf and stem extract of Sorbaria tomentosa. showed the highest inhibition of seed germination at 7500 ppm and stimulated seed germination at low concentration (1000 ppm).

Figure 2 Effect of methanol extracts on seed germination at (a) 7500 ppm and (b) 1000 ppm as a function of the incubation period of seeds.

Discussion

Medicinal plant species all over the world have been playing a vital role in drug discovery efforts. The present study involves the evaluation of crude botanical extracts for their potent cytotoxicity in brine shrimp cytotoxicity assay, for herbicidal or growth stimulation activity by radish seed phytotoxicity assay, and for inhibition of tumor formation in antitumor potato disc assay.

Brine shrimp lethality assay has been considered as a prescreening assay for antimicrobial, antitumor, antimalarial, antifungal, and insecticidal activities. Four of the seven extracts presented significant cytotoxicity. The highest rate of lethality to brine shrimp was observed in the case of leaf and flower extract of Artemisia fragrans.. A number of previous studies indicated potent cytotoxicity in the case of methanol extracts of several plant species. Kanegusuku et al. (2001) reported cytotoxicities of methanol extracts and ethyl acetate fraction from Rubus imperialis. C. (Rosaceae). In another study from Brazil, 60 medicinal plant species were evaluated for their cytotoxicity to brine shrimp (Maria et al., 2000). Only 10% of the species presented ED₅₀ < 1000 ppm. Jacques et al. (2003) demonstrated the screening of 226 methanol and water extracts for lethality toward larvae of brine shrimp and identified several cytotoxic plant species.

Antitumor potato disc assay is a valuable tool that indicates antitumor activity of test compounds by their inhibition of formation of characteristic crown galls induced in wounded potato tissues by A. tumefaciens.. The present study revealed a moderate-to-high rate of inhibition of tumor formation in the case of all methanol extracts tested (Table 3). Leaf and stem extract of S. tomentosa. presented significant inhibition of tumor formation, however, this extract also slightly affected the viability of the A. tumefaciens. strain. Turker and Camper (2002) screened the biological activity of common mullein by antitumor potato disc assay, but no effect on the viability of the A. tumefaciens. strain was observed. Coker et al. (2002) indicated antitumor potato disc assay as an acceptable tool to primarily screen antineoplastic activity of various crude extracts as well as purified fractions regardless of the mode of inhibitory action on tumor formation.

Radish seeds have been used in general toxicity studies because of their sensitivity to phytotoxic compounds (Einhellig & Rasmussen, 1978) and are a standard assay in allelopathic studies (Patterson, 1986). All extracts exhibited toxicity in radish seed bioassay at high doses. A very interesting feature of the present study is the growth-stimulation effect of three extracts (leaf and flower extract of S. nubicola., leaf and stem extract of H. nepalensis., and stem extract of S. nubicola.) at a low concentration (1000 ppm). Ali et al. (2005) also revealed the growth-stimulation effect of one of the fractions (3α.-hydroxy-20-oxo-30-norlupane) of S. nubicola. at 5 ppm in a Lemna. minor phytotoxicity assay. Similar types of results were obtained by Tsao et al. (2002). They studied different fractions of Ailanthus altissima. and observed a growth-stimulation effect at low doses and a growth inhibitory effect at higher doses.

Conclusion

Crude botanical extracts evaluated in the present study can be fractionated in the future and can lead to the discovery of important chemotherapeutic agents. Results of the present study indicate a positive correlation between three assays. A correlation between brine shrimp lethality assay and antitumor potato disc assay has been reported previously. Four of our seven extracts (leaf and stem extract of Acer oblongifolium., leaf and stem extract of Hedera nepalensis., leaf and flower extract of Artimisia fragrans., and root extract of Artimisia fragrans.) presented significant ED₅₀ value in brine shrimp lethality assay and a significant percentage of tumor inhibition in potato disc assay. All extracts used in the present study showed inhibition of root length and seed germination in radish seed phytotoxicity assay as well. These results can lead to the discovery of new anticancer drugs in the future. An interesting aspect of the present study is the low or no inhibition of growth in radish seed bioassay at low concentrations and antitumor activity in the case of three plant extracts. The combined effects of these extracts can be utilized to control crown gall disease in plants.

Acknowledgments

The authors are grateful to Professor Staton Gelvin, Department of Biological Sciences, Purdue University, West Lafayette, IN, USA for providing A. tumefaciens. wild-type strains to our Laboratory for antitumor potato disc assay and to the H.E.C (Higher Education Commission) of Pakistan for providing funds throughout the research project.