Toxicity assessment and phytochemical analysis of Broussonetia papyrifera and Lantana camara: Two notorious invasive plant species

J. Bio. & Env. Sci. 2014 

Journal of Biodiversity and Environmental Sciences (JBES) 

ISSN: 2220-6663 (Print) 2222-3045 (Online) 

Vol. 5, No. 2, p. 508-517, 2014 

http://www.innspub.net 

RESEARCH PAPER 

OPEN ACCESS 

Toxicity assessment and phytochemical analysis of 

Broussonetia papyrifera and Lantana camara: Two notorious 

invasive plant species 

Huma Qureshi, Muhammad Arshad and Yamin Bibi 

Department of Botany, PMAS-Arid Agriculture University, Rawalpindi, Pakistan 

Article published on August 26, 2014 

Key words: Toxicity, invasive species, novel weapons hypothesis, dose response, pharmaceutical drugs, 

agricultural chemicals. 

Abstract 

The blind dependence on synthetics is over and people are returning to the naturals with hope of safety and 

security. As concern regarding the health issues of synthetic medicinal, industrial and agricultural chemicals 

increases, attention is focused on finding some alternative management strategies. Plant derived toxic chemicals 

being comparatively safer hold great prospects in this regard. In the present investigation, polar and nonpolar 

fractions of Brousssonetia papyrifera and Lantana camara were assessed for brine shrimp cytotoxicity, 

sandwich method and radish seed phytotoxicity in search of potential bioactive botanicals. L. camara methanol 

extract (LCME) with LD50


Introduction 

The capability of plants to serve human beings in a 

range of aspects has been well documented since 

antiquity. Nature has produced wonderfully complex 

molecules in the form of secondary metabolites in 

plants that no synthetic chemist could ever dream up 

(Kumar et al. 2011) that have long been and will 

continue to be important sources and models for 

medicinal, agricultural and other industrial raw 

materials (Morris, 1999). Nowadays, people are more 

interested to utilize eco-friendly and bio-friendly 

plant based products (Bibi et al., 2011). The search for 

such bioactive constituents has been quite productive 

in toxic plants (Rates, 2001). If explored fully and 

planned wisely, toxic compounds of plant origin elicit 

quite effective applications in managing health and 

improving the productivity of agricultural systems 

(Khanh et al., 2005; Albuquerque et al., 2011; Farooq 

et al, 2013). 

Phytotoxins are considered a potential source of 

pharmaceutical drugs. Pharmacology is simply 

toxicology at a higher concentration and toxicology is 

simply pharmacology at a lower concentration, 

showing that dose adjustment differentiates a poison 

and a remedy (Parasuraman, 2011). Phytoxins are 

also used as agricultural chemicals such as bioherbicides, 

bio-insecticides, bio-fungicides and biorodenticides 

the necessity of which is due to the 

emergence of resistance to older synthetic molecules 

and their hazardous effects on environment (Awan et 

al., 2012). 

Novel weapons hypothesis for plant invasion states 

that an invader adds toxic chemical(s) to the 

environment that exert strong toxic effects against 

native residents of the exotic range (Chengxu et al., 

2011; Qureshi et al., 2014) but most invasive species 

have been neglected and much less surveyed for 

biologically active chemicals. For this study, two 

invasive species (Lantana camara and Broussonetia 

papyrifera) were selected being declared invasive in 

Pakistan. 

L. camara is evergreen aromatic shrub in family 

Verbenaceae. Its toxic chemicals are reported to be 

present in all parts of the shrub which on release in 

surrounding interfere with many species (Choyal and 

Sharma, 2011). The toxicity of L. camara shrub is well 

known that has long been investigated for 

nematicidal, termiticidal, insecticidal and repellent 

activity (Verma and Verma, 2006; Mohamed and 

Abdelgaleil, 2008; Kalita and Bhola, 2013). On the 

contrary, L. camara has been reported to possess a 

number of medicinal properties to treat various 

human ailments such as malaria, dermatological and 

gastrointestinal diseases, tetanus, tumors and cancer 

(Kalita et al., 2012). 

B. papyrifera is a deciduous tree in Moraceae family. 

Its invaded areas are reported to be with changed 

vegetation patterns considerably having a lower 

diversity of herbaceous as well as woody species 

(Malik and Husain, 2007). Pollens from its flowers 

are allergens causing rhinitis and asthma (Hsu et al., 

2008). B. papyrifera has been used for the treatment 

of dysentery, hernias, oedema, tinea and in 

traditional Chinese medicine (Xu et al., 2010). Its leaf 

extract is reported to be antifungal, antioxidant and 

antihepatotoxic (Yang et al., 2014). 

In the present study the toxicity levels of polar and 

nonpolar extracts of both species were investigated in 

laboratory with the objectives to explore their 

potential as sources of chemicals for medicinal 

(particularly anticancer) and/or agricultural (bioherbicide, 

bio-pesticide) use in the future. 

Materials and methods 

Collection and extraction of plant material 

The plant material was collected from PMAS-Arid 

Agriculture University, Rawalpindi, Pakistan. The 

fresh, healthy aerial parts of both species were 

collected and washed with clean water. Specimens 

were then dried in laboratory at room temperature. 

The dried specimens were ground to a fine texture 

and were separately macerated in methanol and 

chloroform in round bottom flasks for seven days 

509 | Qureshi et al.


followed by filtration. The extracts were concentrated 

using laboratory vacuum rotary evaporator at 40°C. 

The extracts were weighed, labeled and stored at 4°C 

till further analysis. 

Cytotoxic potential of two invasive species 

Brine shrimp lethality assay. Cytotoxic potential 

was investigated by brine shrimp lethality assay 

according to the protocol of Rehman et al. (2005). 

Brine shrimp eggs were hatched into a small 

partitioned tank containing artificial sea water 

(38g/L, pH=8.5). Brine shrimp nauplii with second 

instar stage were used to perform the assay. 

For this experiment, each extract in three 

concentrations (1000, 100 and 10 ppm) was taken 

into small sterile vials in triplicate (9-vials/extract). 

Ten shrimps were added to each vial using Pasteur‟s 

pipette. The vials were maintained under illumination 

at room temperature and survivors were counted 

after 24 h. The resulting data were analyzed by using 

formula; 

Data were evaluated by probit analysis (LdP Line 

Software) to determine the „Lethality Dose 50‟ (LC50) 

at 95% confidence intervals. 

Phytotoxic potential of two invasive species 

Radish seed germination assay. The assay was 

performed as described by Turker and Camper 

(2002). Two types of determinations were carried 

out: 

(a) Detremination of Root length inhibition 

Radish seeds were sterilized with 1% mercuric 

chloride. The Whatman No.1 filter paper was placed 

in Petri plates and 5 ml for each extract 

concentration (10, 100, 500, 1000 and 10000 ppm) 

was added separately. 5 ml distilled water was added 

after solvent evaporation and then ten radish seeds 

were placed in each petri plate followed by tight 

sealing and incubation at 23±2°C. Root length was 

measured after 1, 3 and 5 days. Percentage growth 

inhibition by extracts was estimated using relation 

% Growth Inhibition=100 (PC-PT) /PC 

Where PT and PC represent root length of the 

treatment and control respectively. 

(b) Determination of germination index This 

part of the determination was similar to that of 

earlier determination except for the extract 

concentrations and the number of seeds. Here, three 

different concentrations (100, 1000 and 10000 ppm) 

and 100 radish seeds were used. Germinated seeds 

were counted daily up to 5 days. Germination index 

was calculated as 

Where N1, N2, N3-----Nn=Proportion of seeds which 

germinated on day 1-----n. Each experiment was 

carried out three times. Results were expressed as 

the means of three replicates ± the standard 

deviation of triplicate analysis. 

Sandwich method. Phytotoxicity of plant leachates 

was determined by Sandwich method following the 

protocol of Fujii et al. (2004). Agar solution (0.5% 

w/v) was prepared and autoclaved at 121°C for 15 

min. Plant material was carefully weighed (10, 30, 

50mg) and gently tipped into the wells of a six well 

multi-well plate. By using a pipette, first layer of agar 

(5 ml) was applied, dried plant material rose up that 

was allowed to gelatinize, on top of which a second 

layer of agar was applied. In each dish, five seeds of 

lettuce were placed above agar. Multi dishes were 

covered with aluminum foil to protect them from 

light and kept in incubator at room temperature. 

Length of radicle and hypocotyl was noted after 72 h 

for each plant. Percentage growth inhibition in root 

and hypocotyl in each treatment was estimated using 

the equation 

% Growth Inhibition=100 (PC-PT) /PC 

Where PT and PC represent root/hypocotyl length of 

the treatment and control respectively. 



Qualitative phytochemical analysis of plant extracts 

Test for alkaloids. Mayer‟s reagent: Mercuric 

chloride (0.356 g) was dissolved in 60 ml of water and 

potassium iodide (5g) was dissolved in 20 ml water. 

Both solutions were mixed and volume was made up 

Test for phenols. Plant extracts were treated with 

3-4 drops of freshly prepared FeCl3 solution. 

Appearance of bluish black color indicated the 

presence of phenols. 

to 1000 ml with distilled water. 

Test for saponins. Plant extract (0.5 g) was 

Dragendorff‟s reagent: Solution A: Basic Bismuth 

nitrate (1.7 g) and tartaric acid (20 g) was dissolved in 

80 ml of distilled water. 

dissolved in boiling water in a test tube, allowed to 

cool and shaken thoroughly. Froth formation was 

observed to indicate the presence of saponins. Test 

for tannins. Plant extract (0.5g) was boiled in 20 ml 

Solution B: Potassium iodide (16 g) was dissolved in 

40 ml of distilled water. 

of distilled water in a test tube and filtered. 0.1% 

FeCl3 was added to the filtrate. Appearance of 

brownish green or blue black coloration showed the 

Solution A and B was mixed in 1:1ratio. Plant extract presence of tannins. 

(0.5 g) was mixed with 8 ml of 1% HCl, warmed and 

filtered. Filtrate was treated separately with Mayer‟s 

reagent and Dragendorff‟s reagent. Turbidity or 

precipitation was observed to indicate the presence of 

alkaloids. 

Test for glycosides. Extract (0.5 g) was dissolved 

in 2.0 ml of glacial acetic acid containing one drop of 

0.1% FeCl3 solution and was then underlaid with 1.0 

ml of concentrated H2SO4. A brown ring at the 

interface indicated the presence of glycosides. 

Test for flavonoids. Plant extract (0.5 g) was 

shaken with pet. ether to remove the fatty materials. 

The defatted residue was dissolved in 20 ml of 80% 

ethanol and filtered. Filtrate was mixed with 4 ml of 

1% KOH. A dark yellow color was observed to indicate 

the presence of flavonoids. 

Results 

LCMA showed cytotoxicity with median lethality dose 

of 354.27 ppm and 23.33% lethality at a concentration 

of 10 ppm. LCCE and BPME indicated weak cytotoxic 

activity with 50% lethality at 1000 ppm while BPCE 

was found not toxic with 40% lethality at 1000 ppm 

Test for coumarins. Plant extract (0.5 g) was taken (Table 2). So, it is evident that LCME has cytotoxic 

in a small test tube and covered with filter paper activity which can further be exploited for 

moistened with 1 N NaOH. The test tube was placed 

in boiling water for few minutes. The filter paper was 

removed and examined in UV light for yellow 

pharmacological activity because brine shrimp is 

considered as a suitable probe for screening the 

pharmacological activities in plant extracts. 

fluorescence to indicate the presence of coumarins. 

Table 1. Summary of plants, parts used, solvent used and extraction method. 

Plant Species 

Family 

Vernacular 

name 

Part used 

Solvent 

used 

Extraction 

method 

Extract 

obtained 

Broussonetia papyrifera 

(L.) L‟Her. ex Vent. 

(Syn. Papyrius 

papyrifera, Morus 

papyrifera) 

Moraceae 

Jangli 

Shahtoot 

Aerial parts 

(350g) 

Aerial parts 

(420g) 

Methanol 

Chloroform 

Cold 

maceration 

Cold 

maceration 

24g 

25.5g 

Lantana camara L. 

(Syn. Camara vulgaris, 

Lantana scabrida) 

Verbenaceae 

Panj Phuli 

Aerial parts 

(290g) 

Aerial parts 

(350g) 

Methanol 

Chloroform 

Cold 

maceration 

Cold 

maceration 

21g 

20.5 



Table 2. In vivo cytotoxicity of L. camara and B. papyrifera extracts on Brine shrimp nauplii. 

Plant Extract 

Concentration 

(ppm) 

Surviving 

Dead nauplii 

after 24 hrs. 

nauplii after 24 

hrs. 

R1 R2 R3 R1 R2 R3 

Total 

Survivors 

Mortality 

(%) 

B. papyrifera 

(Met.) 10 1 1 1 9 9 9 27 10.00 1000 

100 3 2 3 7 8 7 22 26.67 

1000 5 5 5 5 5 5 15 50.00 

B. papyrifera 

(Chl.) 10 2 3 3 8 7 7 22 26.67 >1000 

100 3 3 5 7 7 5 19 36.67 

1000 4 4 4 6 6 6 18 40.00 

L. camara 

(Met.) 10 1 4 2 9 6 8 23 23.33 354.27 

100 4 4 3 6 6 7 19 36.67 

1000 6 7 5 4 3 5 12 60.00 

L. camara 

(Chl.) 10 1 1 2 9 9 8 26 13.33 1000 

100 2 3 3 8 7 7 22 26.67 

1000 4 6 5 6 4 5 15 50.00 

LD50 

The effect of five different concentrations (10000, 

1000, 500, 100 and 10 ppm) of the extracts on root 

growth inhibition of radish seedling indicated highest 

percentage inhibition by LCME (72.85±2.69%) at 

10000ppm. All extracts inhibited root growth at 

10,000 ppm (Fig. 1). The effect of three different 

concentrations of each extract (100, 1000 and 10000 

ppm) on seed germination was also observed and a 

gradual decrease in seed germination index for all 

extracts was observed until the fifth day of incubation 

with maximum inhibition at 10000 ppm (Fig. 2). 

BPCE showed most pronounced decrease in index of 

seed germination at 10000 ppm (63.13±5.86%). In all 

extracts, both the parameters were found to be 

directly correlated with concentration of extract used. 

However, seed germination velocity was less affected 

as compared to root length. This may be due to the 

reason that seeds are protected by their integuments, 

so they seem to be less sensitive to phytotoxins than 

seedlings (Dandelot et al., 2008). 

Fig. 1. Radish root inhibition (%) by L. camara and B. papyrifera extracts. 



Fig. 2. Radish seed germination index by application of L. camara and B. papyrifera extracts. 

Exposure of lettuce seeds to both invasive species has 

registered growth inhibition in roots and hypocotyles. 

Inhibition of root and hypocotyl length in lettuce 

seedlings was influenced by the concentration of plant 

material. Maximum toxic potential on root inhibition 

has shown by L. camara dried sample at 50mg 

concentration (49.26±5.40%). At the same 

concentration B. papyrifera showed inhibition of 

42.94±1.32% (Fig. 3). In the same vein maximum 

toxic potential on hypocotyl inhibition in lettuce 

seedlings was predicted by B. payrifera 50mg sample 

(32.15±2.18 %) while L. camara showed 25.90±1.04% 

inhibition at the same concentration. 

Preliminary phytochemical analysis indicated that 

alkaloids, saponins, tannins, glycosides, coumarins, 

flavonoids and phenols were present in LCME and 

BPME. Saponins were found to be absent in LCCE 

while tannins and saponins were not indicated in 

BPCE (Table 3). 

Table 3. Preliminary phytochemical analysis of plant extracts. 

Plant Extract Alkaloides Flavonoids Saponins Tannins Coumarins Glycosides Phenols 

LCME +++ +++ ++ ++ + ++ +++ 

LCCE ++ + - + + + ++ 

BPME +++ ++ +++ + + ++ +++ 

BPCE ++ + - - ++ + + 

„+‟ weak „++‟ moderate „+++‟ strong presence „-„absence 

Discussion 

With direct or indirect utilization capability in 

medicinal, agricultural and industrial raw materials, 

study of phytotoxins serve society to protect humans 

and the environment from the deleterious effects of 

synthetic toxicants. The Novel Weapons Hypothesis 

of plant invasion holds that invader plants release 

some toxic substances that may be synthesized in any 

Fig. 3. Root and hypocotyl reduction (%) in lettuce 

seedlings by L. camara and B. papyrifera. 

plant part, but leaves are considered to be most 

consistent producers of these phytotoxins (Umer et 

al., 2010). In this scenario, we investigated toxicity 

levels of polar and nonpolar extracts from aerial parts 



of two species declared invasive in Pakistan through 

prescreening dose response bio-assays (brine shrimp 

cytotoxicity assay, sandwich method and radish seed 

phytotoxicity assay) and qualitative phytochemical 

analysis was performed to determine possible 

secondary metabolites accountable for toxicity. 

The brine shrimp lethality assay is used to evaluate a 

broad spectrum of pharmacological activities of 

natural substances taking into account the basic 

premise that toxicology is simply pharmacology at a 

lower dose. From pharmacological point of view, a 

good relationship has been found with the brine 

shrimp lethality and antitumor activity of plant 

extracts (Pour and Sasidharan, 2011). Out of four 

extracts, cytotoxicity was shown by LCME (LD50 = 

354.27ppm) while the other extracts showed 

LD50≥1000ppm that is not significant. Cytotoxicity 

of this plant is also reported earlier in cell line 

experiments (Raghu et al., 2004 and Pour et al., 

2011). The observed lethality of this extract to brine 

shrimps indicates the presence of cytotoxic and 

probably antitumor components in this plant. 

investigating allelopathic effect of Amomum 

krervanh Pierre ex Gagnep against five test plant 

species; Digitaria sanguinalis L., Lactuca sativa L., 

Lepidum sativum L., Medicago sativa L. and Phleum 

pratense L. 

Phytotoxicity assessment through sandwich method 

revealed maximum root inhibition by 50mg 

pulverized sample of L. camara (49.26±5.40%) while 

maximum hypocotyl inhibition by 50mg B. 

papyrifera (32.15±2.18%). These findings support 

more root sensitivity to phytotoxins in comparison to 

hypocotyl. Relative greater root sensivity may be 

explained by the fact that after the seed germination, 

roots are the first to come in direct contact with toxic 

chemicals (Khaliq et al., 2013). Parallel results were 

forwarded by Fujii and Aziz (2005) while examining 

the phytotoxic effect of the extracts from 14 plant 

species of plain areas on the growth of lettuce seeds. 

Anjum et al. (2010) evaluated inhibitory effect of 14 

medicinal plants through sandwich technique and 

suggested strong inhibitory effects of Albizia lebbeck 

and Broussonetia papyrifera. 

Determination of phytotoxicity of a plant species 

helps in the formulation of natural plant growth 

regulators or biological herbicides (Khan et al., 2011). 

The radish seed germination assay is a valuable tool 

that indicates general phytotoxicity because of their 

sensitivity to toxic compounds. All extracts exhibited 

phytotoxicity in radish seed bioassay at high dose 

maximum inhibition was shown by LCME 

(72.85±2.69%). Proportional inhibitory effects of 

concentration were also reported by Mahmood et al. 

(2010) while investigating the phytotoxic potential of 

Sorghum bicolor (sorghum), Helianthus annuus 

(sunflower), Brassica napus (brassica), Zea mays 

(maize), Oryza sativa (rice) and Morus alba 

(mulberry) water extracts suppressing germination 

and growth of horse purslane in the laboratory 

bioassay. Khan et al. (2011) also revealed dose 

dependent phytotoxic effects of methanol extracts of 

thirteen medicinal plants. Dose effect was also 

reported by Pukclai and Kato-Noguchi (2012) while 

Plants owe their bioactivities by the presence of 

certain biochemicals. Chemicals that impose toxic 

influence are called phytotoxins that are classified as 

secondary plant metabolites (Hadacek, 2002) 

belonging to diverse chemical groups. Alkaloids, 

cumarines, flavonoids, hydroxamic acids and 

phenolic acids are generally cited phtotoxins 

responsible for their toxic activities in ecosystem. The 

phenolic compounds (such as flavonoids, tannins and 

phenols) are reported as the most common and 

widely distributed toxic metabolites in plants 

(Arowosegbe et al., 2012). Similarly, various studies 

indicated that different phytochemicals cause 

cytotoxicity/cell damage. Alkaloides, flavonoids, 

tannins and other phytochemicals produce cyototxic 

effects on tumors (Sharma et al., 2011). Toxicity of 

plants may be induced by one of these biochemical or 

different biochemicals may act in synergism to induce 

toxicity (Hussain and Reigosa, 2011). Thus, the 

toxicity of extracts in this work could be ascribed to 



the toxic compounds such as phenolic compounds, 

alkaloids and saponins that were found to be present 

in the extracts. Whatever may be the toxic principle, 

the promising result displayed by the polar plant 

extracts in the cytotoxicity and phytotoxicity assays 

justified the efficacy of these plants as a potential 

source of pharmaceutical, industrial and agrochemical. 

Conclusion 

In conclusion, this study demonstrated that L. 

camara and B. papyrifera have bioactive toxic 

principles. LCME has cyotoxic activity towards brine 

shrimps and comparatively high phytotoxic ability. 

Thus, we consider that preferentially this plant might 

have some useful influences in two fields i.e. medicine 

and agriculture. However, further toxicity studies are 

needed for dose adjustment and for isolation and 

structure elucidation of bioactive compounds 

responsible for the observed toxicity. 

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Toxicity assessment and phytochemical analysis of Broussonetia papyrifera and Lantana camara: Two notorious invasive plant species

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