Acute toxicity, cytotoxic, phytotoxic, muscle relaxant, analgesic, antispasmodic and antimicrobial potential of Cocculus pendulus

The present experimental studies describes the acute toxic, cytotoxic, phytotoxic potential, analgesic, GIT motility and antimicrobial activity of Cocculus pendulus. The pharmacological profile has been evaluated by conducting acute toxic, cytotoxic, phytotoxic potential, analgesic, GIT motility and antimicrobial potentials. The plant is unsafe for consumption at higher doses, while safe at lower doses. The plant showed significant cytotoxic potential of Cocculus pendulus crude stem extract (CSE) with 7.82 LD50 value and Cocculus pendulus crude root extract (CRE) with 4.18 LD50 value. Phytotoxic potential was also significant (CSE) with 32.32 FI50 value and (CRE) with 24.68 FI50 value. Muscle relaxant effect was significant at 90mg/kg both in CSE and CRE in traction and inclined tests which were 70%, 85%, 75% and 80% respectively. Analgesic activity were carried out via acetic acid induced writhing method while antispasmodic activity was done through charcoal motility method. C. pendulus stem extract (CSE), C. pendulus root extract (CRE) and isolated fractions showed highly significant (**P<0.01) analgesic effect at higher doses. CSE and CRE and isolated fractions at all doses reduced the motility of GIT in albino mice. CSE, CRE and their isolated fractions showed remarkable reduction in the zone amplitude against Proteus sp and Xanthomonas sp. These effects of crude and isolated fractions justify its use in folkloric medicines for cytotoxic, herbicidal, pain relief and various gastro-intestinal disorders.

the bark of stem bark and root for the same purpose [3]. In Senegal the Toucouleur and Paul people use stem and root bark decoctions against intestinal parasites and gonorrhea [1]. The root has a great reputation in Senegal against biliousness and menstrual problems and as a diuretic [3]. It is also a part of medicines against jaundice, yellow fever, leprosy, syphilis and as an aphrodisiac in the area [4]. It has been recorded that many medicinal plants have been used for curing many diseases as traditional treatments for thousands of years throughout the world. In less developed countries especially in rural areas, the peoples use medicinal plants as primary source for treatment [5]. In developing countries about 80% of the people are dependent on herbal plants for their primary health care [6].
In the present study Cocculus pendulus has been examined against cytotoxic, phytotoxic, acute toxic, muscle relaxant, analgesic, antispasmodic and antimicrobial bioassays.

Materials and methods Collection and preservation of the plant
The fresh stem and root of the experimental plant were collected in January 2015 from the Tribal area, Sama Bada Bera, Labi Khel, F.R Peshawar. Each specimen was cleaned and washed. Both stem and root were completely dried under the sun. Both specimens were grinded. After grinding the powdered drugs were kept in impermeable bottles and were used for different pharmacological bioassays. Preparation of extract Dried powder of stem and root of the plant were extracted with ethanol kept on rotary shaker for 48 hours. After that it was filtered, the filtrated solution were collected were processed in rotary to make the final volume 1/5 of the original volume and stored in impermeable bottles at 4 o C for further processing [7]. Experimental animals Albino mice were used in all bioassays. Animals were purchased from National institute of health (NIH) Islamabad. The animals were provided with favorable laboratory conditions (25˚C) and were provided with standard food and water.

Isolation of alkaloids fractions
The methodology of [8] was adopted for the quantitative screening of total alkaloids. 100 ml of acetic acid (10%) was taken in which 2g crude ethanolic extract of stem and root were added. The solution was allowed to stand for 4 hours and then filtered. After filtration, the extracts were placed on a water bath for further concentration to reduce the volume to one-fourth. Precipitate formation occurred by the addition of Concentrated NH4OH drop wise. Dilute NH4OH was used for washing the collected precipitate. The obtained product was collected and dried.

Isolation of flavonoids fraction
Quantitative determination of flavonoids was carried out following the methodology of [9]. 2g crude extract in 20 ml hot distilled water of both root and stem were dissolved to make a solution in a beaker. The solution was then filtered and placed in refrigerator for 3 hours. 10 ml ethyl acetate was added to the solution after refrigeration. The flavonoids become precipitated and were filtered. The filtrate was collected and dried. Acute toxic activity The stem (CSE) and root (CRE) crude ethanolic extract were subjected for the acute toxicity bioassay. Albino mice of either sex weighting 20 -25 grams were selected for bioassay. Seven groups of the animals were made. Each group was provided with 4 animals. Before performing the experiment, the animals were accustomed with the laboratory conditions. The control group were provided with normal saline and the remaining groups were provided the crude ethanolic extract of CSE (50, 70 and 90mg/kg) and CRE (50, 70 and 90mg/kg). The animals were continuously observed that either the extract show toxicity or safe for consumption [10].

Cytotoxic bioassay
The methodology of [11] was carried out to determine cytotoxic potential of the crude extracts. Cytotoxic bioassay has been conducted using Brine shrimp's lethality bioassay. Brine shrimp's eggs were kept in a container. The container were divided into two unequal parts contain 3.8% sea salt solution by a perforated septum. Brine shrimp's eggs were sprinkled in the smaller part of the container and was covered with black paper in order to create darkness, while the larger part of the tank was illuminated with electronic bulb. After two days of incubation, the eggs hatched and the nauplii start swimming towards the illuminated part of the container. 15mg crude ethanolic extract were dissolved in 1.5 ml distilled water and stock solution were prepared. From the stock solution varying concentrations of 5, 50 and 500 μg/ml were taken, equivalent to 10, 100 and 1000 μg/ml. 3 test tubes were taken for each concentration. Each test tube was provided with 10 brine shrimp larvae containing 5 ml saline solution. After 24 hours the number of alive shrimp's were counted in each test tube with the help of magnifying glass. The percentage mortality data were calculated and the 50% lethal dose (LD50) values were recorded [12]. Phytotoxic activity The methodology of [11] was conducted for phytotoxic bioassay using Lemna minor for experiment. 20mg of the extract was dissolved in 2ml water from which different concentrations 10, 100, 1000 μg/ml were made. For each concentration 3 petri dishes were taken. Each petri dish was provided with 20 ml E-medium. Other petri dishes (3 petri dishes for each) were supplied with Emedium taken as negative control and standard drug Atrazine taken as +ve control. 10 plants containing 3 fronds were kept under 12 hours day light in petri dishes. The fronds were observed regularly and their number were counted after three days. The following formula were used for recording the % growth inhibition [10].
Inhibition % = (100 − Number of fronds in test sample) (Number of fronds in negative control) × 100

Muscle relaxant activity Materials required
Diazepam, distilled water, albino mice, rigid wire, inclined plane and selected parts crude extracts and isolated alkaloids and flavonoids fractions from the crude extracts.

Traction test
The experiment was conducted on a metal wire. The wire was fixed tightly in between two stands about 60 cm above the table. The animals were divided into control and test groups each with 4 animals. Group I was treated with normal saline (negative control) and group II was provided with diazepam (positive control) at (10 ml/kg) and (1mg/kg) doses respectively. Rest of the groups were treated with CSE (50, 70 and 90mg/kg) and CRE (50, 70 and 90mg/kg) respectively. All the animals were freely hanged by their hind legs after 30 minutes of the treatments and hanging time was recorded for 5 seconds.
Hanging less than 5 seconds reflects the presence of muscle relaxant property otherwise absent [13].

Inclined plane test
The animals were divided into control and test groups each with 4 animals. Group I was treated with normal saline (negative control) and group II was provided with diazepam (positive control) at (10 ml/kg) and (1mg/kg) doses respectively. Rest of the groups were treated with CSE (50, 70 and 90mg/kg) and CRE (50, 70 and 90mg/kg) respectively. Each group was left on inclined screen and observed whether the effect of doses was significant to cause the mice to slide down of the screen or non-significant and the mice remained on the inclined slope [13].

Analgesic bioassay Acetic acid induced writhing test
Analgesic activity was conducted on albino mice of either sex having 20-25 g weight. Animal were distributed into 20 groups (n=4). Diclofenac sodium (10 mg/kg) was given to Group-I and normal saline (10 ml/kg) were given to Group-II and other groups were treated with CSE (50, 70 and 90mg/kg) and its isolated Alkaloids from crude stem extract (ACSE) ( Fig. 2).

Antibacterial bioassay
The stem and root crude ethanolic extracts and their isolated fractions of C. pendulus were assessed using agar disc diffusion method for antimicrobial activity. The stem and its isolated fractions at all doses showed remarkable results. The highest zone amplitude reduction was noted against Xanthomonas sp (17mm) by CSE, while ACSE produced (18mm) against Xanthomonas sp and FCSE produced (18mm) against Proteus sp. Similarly, the root and its isolated fractions also showed remarkable zones of inhibition at higher doses. The highest zone amplitude reduction (18mm) was noted against Proteus sp, while ACRE produced (18mm) zone of inhibition against Proteus sp and FCRE produced (18mm) against Xanthomonas sp (Table 9; Fig. 9).

Discussion
The above results of acute toxic bioassay revealed that this plant is not safe for the consumption at higher doses, while safe at lower doses as discussed above. Brine shrimp cytotoxic bioassay a simple, economical and efficient method of screening of test articles for anti-cancer potential. When CSE and CRE were tested against anti-cancer potential both the extracts showed significant anticancer potential. This indicate that this plant is good for the therapy of cancer. Weeds interference noticeably responsible for the huge economic loss to the quality and quantity of agricultural crops all over the world. It is estimated in US that weeds cause a loss of at least 12% costing to nearly US$ 33 billion while the situation is more alarming in developing countries [18]. As an agriculture country, Pakistan yielding high quality and quantity of various cereals and crops. Large quantity of these crops may be damaged due to poor weed control strategies.
In agricultural sectors, synthetic herbicides are widely used for the weed control. However, various factors such as water and soil pollution, herbicide residues, detrimental effects and herbicide-resistant weed populations restricted the use of synthetic herbicides. The degree of harm caused by insects and diseases are less than that cause by uncontrolled weeds, but its effects are unseen. Due to competition for sunlight, water and fertilizer, they also reduce the crops yield. Furthermore, weeds are actively involved in habitat provision for insects which help in spreading of disease. So, for increasing the production of various crops, weeds controlling is very essential. In our present study both the extracts showed significant phytotoxic potential, which indicate that this plant has good weedicidal potential [18]. Traction and inclined plane tests are techniques use for the determination of muscle relaxant properties in animals. The animals in these models can spend time on wire and inclined plane, less time spent more indicates a muscle relaxant effect of a tested material. Our findings reveal that crude ethanolic extract of both stem and root exhibit significant activity. So, it is clearly indicated the muscle relaxant active substances (secondary metabolites) of the plant are concentrated in these two extracts.
In addition, our results are well in lineage with the standard drug diazepam used in the study Analgesic potential of medicinal plants can be determined by using Acetic acid-induced writhing method [19]. In acetic acid model pain sensation is caused by the production of inflammation causing factors arachidonic acid from phospholipids tissues through an enzyme known as cyclo-oxygenase (COX), that release prostaglandin specifically PGE2 and PGF2α. In peritoneal fluids, the level of lipoxygenase also become up which increases capillary permeability [20]. The substance preventing the writhing by reducing pain due to inhibiting prostaglandin synthesis, this is peripheral analgesic effect [19]. In the current experiment, the results of crude extract and fractions showed remarkable reduction of writhing reflux. These observations strongly suggested that C. pendulus and its fractions has good analgesic potential.

Various other investigators like [3, 21-27]
carried out similar activity on several medicinal plant from various families like Hyptis suaveolens, Artocarpus lakoocha, Aphanamixis polystachya, Kalanchoe pinnata, Justicia prostrata, Catharanthus roseus and Skimmea. Laureola reported a good significant activity of these plants on mice and various other test using different test and methods. The workers suggested that the analgesic effect of these is due the presence of secondary phytochemical especially flavonoids and alkaloids use for analgesia. Hence, these all workers strongly supported and in line with our research because in C. pendulas the alkaloids and flavonoids good effect as compared to crude extracts, hence it is suggested that the specific alkaloids and flavonoids responsible for treatment of analgesia should be determined, characterized, isolated and should be used instead of haphazard synthetic and expansive drugs as natural, economic, easily available and safe source medicines. Diarrhea is a very common ailment and drastic disease in most of the tropical countries of the world causing millions of deaths every year [28]. Along with modern advance synthetic drugs the herbal medicines are mostly effective and economic therapy for diarrhea in several Asian countries. Numerous medicinal plants have been testified to be most effective in safe treatment of diarrhea dysentery with no side effect. C. pendulus stem and root crude extract and isolated fractions showed significant results against diarrhea. In folk medicines, for treatment of gastrointestinal ailments many plants can be used. Nowadays many people also turn to the use of natural drugs for the treatment of intestinal complications. Several other researchers also agree with us as [29-32] reported significant antispasmodic activities various plants like Swertia chirata, Myrtus communis. Manilkara zapota, Cynanchum viminale, Symplocos paniculata, and Withania somnifera in gastrointestinal motility and castor oil induced diarrhea in mice and hence suggested study on medicinal plants provides basis for the vernacular use in gastrointestinal system. We also suggested the C. pendulus as a good safe and natural antispasmodic plant because it shows a significant reduction in custard oil induced diarrhea in mice and suggested it further be advance explored and specific responsible substances must be isolated. Plants extracts are important source of chemotherapeutic and antibiotic agents' due to existence of combination of primary and secondary active and non-active metabolites. The Unani System of Medicine still need to testify plants and their crude extracts according to the recent measures to confirm their active potential. Many reports showed that plant crude drugs with no side effect and reaction have good antibacterial, antifungal and anti-inflammatory potential [33]. Plants chemical are natural antimicrobial agents are considered to be environment friendly used as bio-control agents and easily available [34]. Various pure secondary metabolites and active constituents have been identified, isolated and used as effective natural drug for treatment of dangerous human diseases. The result obtained from present investigation concluded that the crude extract and isolated fractions showed significant results against all bacterial strains. Similar antimicrobial bioassay was conducted by [35] on Bligha sapida against five bacteria and five fungal and reported significant growth inhibitory activity.
[36] Used methanolic extracts and various fractions of Cardiospermum halicacabumon and reported that ethanolic extract showed good results. [37] Worked on Impatiens bicolor [38] used Nephelium lappaceum against pathogenic bacteria and reported similar dose dependent zones of inhibition against bacteria. [39] Proved that Litchi leaf contained luteoline, epicatchin, procyanidin and rutin which are strongest antimicrobial against. All these studies strongly supported our work. Hence it is suggested that C. pendulus may also be used as antimicrobial drug. Therefore, this has prominent perspectives in developing antimicrobial agents through further biological research.

Conclusion
In conclusion, we can say that Cocculus pendulus contain active ingredients possessing cytotoxic, phytotoxic, muscle relaxant, analgesic, antispasmodic and antimicrobial potential. Our present findings provide a way for the exploration of the plant and isolation of the active constituents for further advance studies.

Authors' contributions
Conceived and designed the experiments: B Ullah, Performed the experiments: M Nafees, Analyzed the data: S Ullah, Contributed materials/ analysis/ tools: M Ibrar, Wrote the paper: M Nafees.