Pharmacognostic, Antimicrobial and Toxicological Studies of a Seasonal Medicinal Plant; Tagetes Patula L

Authors’ Contributions 1 Conception & Study Design, Data Collection & Processing, Data Analysis and/or Interpretation, Drafting of Manuscript. 2 Conception & Study Design, Drafting of Manuscript, Critical Review. 3 Drafting of Manuscript, Critical Review. 4 Data Analysis and/or Interpretation, Critical Review.


I N T R O D U CTION
Plant derived products serves as potential source of modern drugs. In developed countries 13% of medicine consumed are based on herbs However, only fewer plants i.e. 15% were undergone proper investigation. According to report generated by World Health Organizations herbal medicines are easier to access, effective, nontoxic, relatively cheap and have lesser side effects [1]. Natural plant products offered great treasure of secondary metabolites which have been displayed enormous biological and pharmacological activities [2] The ornamental plant Tagetespatula (Asteraceae) is well known for its reddish yellow, orange colored beautiful flowers [3]. Name of plant is common as French marigold and Genda [4]. Tagetespatula is rich in commercially important carotene compounds, helenien, xanthophyll, essential oil ,thiophene, steroids and terpenoids [5,6].The plant can be used in the form of extracts, powder, decoction, juices, infusions and oils for the treatment of various diseases.i.e juice of flowers are used in this remedy given to a patient produces strengthening of connective tissues, enhance healing process and decreases inflammation and act as analgesic. Crushed flowers are used as remedy to purify blood in 15 days. Traditionally flowers heads are used to kill internal worms and proved as anthelmintic. Commercially flower oil can be used as insect repellent and modifier in hair lotions also the oil has been used as food additive and food colorants (4). oil also acts as antiseptic as well as antifungal in candidiasis and other infections whereas roots and seeds are employed as purgative. Aqueous extract of the flower showed antibacterial while methanolic extract possessed anti inflammatory activity [5].

M A T E R I A L S A N D M E T H O D S Plant Material
The fresh flowers of T.patula (2 kg) were purchased from local nursery of Karachi, in November 2016 and identified by Prof. Dr. Surraya Khatoon, Department of Botany, Faculty of Science, University of Karachi voucher specimen number (0043) were deposited in herbal museum of Department of Pharmacognosy , University of Karachi.

Preparation of Extracts
Fresh flowers of Tagetes patula were first clean and after that ray and disc florets were removed and percolated in ethanol at room temperature for 10-15 days. After that percolate was filtered by whattman filter paper no 1 and dried in rotary evaporator at 39-40 ºC under reduced pressure. This procedure was repeated thrice. Obtained semisolid ethanolic extract was lyophilized on freeze drying apparatus under reduce pressure, to get powder brown extract (24gm).

Histological Evaluation
The transverse section of the florets of T.patula were examined through cellular sequences by making permanent slides. Method was adopted by staining and glutening. After that complete histology was studied by means of electronic microscope. [7,8].

Microscopic Examination
Flower were first clean to remove dirt and unwanted debris and then dried under shade thereafter dried powder material evenly ground and analyzed for powder microscopy. The powdered material treated one by one with glycerin (50%), iodine solution (5%) and chloralhydrate solution (10%) [7,9].

Solubility and Reaction of Powder Drug with Various Reagents
Tagetes patula showed different solubility patterns and changes of colour when mix up in various chemical agents [10].

Antimicrobial Activity
Microorganisms were obtained in this research work from HEJ,

Antibacterial Activity
The antibacterial activity was evaluated by Well diffusion method. Amoxicillin was used as standard drug and as medium, nutrient agar was employed. Inoculation of nutrient plates were done within 24 hrs. Old bacterial cultures around 10 8 -10-6 CFU/ml were taken. Wells with size of 8 mm were made in the nutrient media. Reference drug along with samples were incorporated into the media with concentration of 100mg/ml. DMSO was used as negative control. The inhibitory zone of Gram+ve and Gram-ve organisms were noted and then compared with standard. [11].

Antifungal Activity
The method used to screen antifungal activity of crude extract of Tagetes patula was Agar dilution. Miconazole, and Amphotericin B were used as reference drug. Sabouraud dextrose agar (SDA) subjected to maintain fungal organism growth. The broth was kept in incubator for 24 hrs. at 37 ºC. A dilution of 1:100 in distilled water was used in the test. The petri plates prepared with SDA. In the media wells (6mm) were dug for test and standard samples each having concentration (100mg/ml) respectively. While for negative control DMSO was used. The plates were remains in incubator for 24 hrs. at 37ºC. [12] Results were noted by measuring zone of inhibition in mm and then compared with standard.
The obtained mean values were calculated as follows: 100-Linear growth in test (mm)/Linear growth in control (mm) x100.

Larvicidal Activity
The toxic manifestations of bioactive compounds depicted some information about high or low level of toxicities by Brine shrimp bioassay method which is a rapid and preliminary method. The brine shrimps eggs were conveniently hatched in the artificial sea water at 25 C in 24 hrs. This was prepared with 3.8 g sea salt in 1000ml of distilled water and thereafter filtered. By this method large number of larvae have been produced. Ethanolic extract in concentration of 10,100 and 1000µg/ml with 5 ml of sea water and ten shrimps in each vial were kept in incubator for 24 hrs. at 27 ºC. The numbers of shrimps survived was counted after 24 hrs. In this protocol, the negative control was served as solvent. While the standard drug Ectoposide with LD50=7.465µg/ml was used as positive control. [13].

R E S U L T S
Cellularprofile of crude drug was examined on the basis of microscopic techniques shown in [ Figure. Table 1.
Results of antibacterial and antifungal activity against various pathogens are shown in (

D I S C U S S I O N
In best of our knowledge powder microscopy and histological studies of T.patula florets was first time conducted in this studies. Histological evaluation revealed that epidermis consist of cuticle and single layered barrel shaped cells. On lower side small cystolyth were present. Upper zone consists of thin walled endodermis. While Middle zone composed of lignified cells but various oil cells abundantly found in lower zone. Parenchyma was wavy with numerous pigments. Both upper and lower layer covered with glandular trichomes. Various oil glands seen in transverse section [ Figure.1]. In powder florets regular bands of red coloring globules were observed. Glandular trichomes were multicellular, unbranched, uniseriate i.e. globular, ovoid head was present with multicellular stalk. Group of oil glands seen in parenchyma of hypanthum. Hair like fibers were abundantly seen. Spherical, spiny pollen grains were present. Rectangular segmented thin layer epidermis was noticed, air spaces were tiny or absent.
[ Figure. 2]. In solubility testing both ray and disc florets showed polar characteristics i.e ray florets were soluble in sulphuric acid but slightly soluble in acetic acid, methanol and water while in Benzene they were insoluble .Whereas disc florets were insoluble in acetic acid and Benzene while soluble in water methanol and sulphuric acid. (Table 1) Ethanolic extract showed good activity against gram positive bacteria i.e. S. aureus and B. subtilus with respective zone of inhibition 12 mm and 20 mm .The flower extract was also effective against 90 % gram negative bacteria (E. coli, S. typhi, P. aeruginosa,) with respective zone of inhibition 17 mm, 16 mm, and 17mm.when compared with standard drug Amoxicillin .It was interesting to note that in our study T.patula extract showed same strong inhibition against E.coli which is in agreement with previous research study [14] No zone of inhibition was shown in case of S. flexanri. Generally gram negative bacteria shown less susceptibility towards plant extracts as compare to gram positive bacteria due to the fact that their outer wall is composed of lipopolysaccharides and lipoprotein which behaves resistant for antibacterial entry ( Table 2). [15]. The ethanolic extract has prominent antifungal activity against human pathogen i.e A. flavus, C. glabrata and especially T. longifusus, with 60% and 70% inhibition respectively. Fungal pathogen produces severe problems around world causing a number of human, plant and animal diseases. Like pathogenic A. flavus which is a real cause of aflatoxins can contaminate food commodities.
T. longifusus (dermatophyte) responsible for hair, nail and scalp. infections [16] profound activity was also shown in case of F.solani (70%) and M. canis (70%).While least antifungal activity was demonstrated in case of C.albicans ( Table 3). Similar outcomes have been reported by other studies which further proved the antimicrobial potentials of this plant [2,17,18,19,] .The floral extract exhibited remarkable larvicidal activity, it was observed at concentration of 10 and 100 µg/ml the mortality rate was found to be 30% and 90% respectively and when the dose was increased up to 1000 µg/ml the rate of death was 100% .Therefore ,it is evident that when dose is increased the extracts works effectively. (Table 4) The strong cytotoxic activity (LC50 5.58 µg/ml) was also confirmed in methanolic leaves extract of same plant by Rahul khudus et al [2].

C O N C L U S I O N
On the basis of this current study it can be concluded that Tagetes species showed good antibacterial and antifungal activities and might be used as a good candidate in healing wounds and other certain kind of skin infectious conditions. Moreover Pharmacognostic standardization has also performed that is necessary for quality control and authentication of the medicinal plant.