An evaluation of phytochemical screening of Lantana camara Linn. (An invasive plant species of Pakistan)

Lantana camara Linn. is an invasive plant species in Pakistan, but also a very important medicinal plant. Current research was conducted to determine its different metabolites. FeCl 2 was used for detection of phenolic compounds and found in different plant parts in higher quantity. For the determination of terpenoids and steriods CHCl 3 and H 2 SO 4 while NaOH was used to test cumarines and quinones and were found in all plant parts in sufficient amount. Distilled water and HCl were used to test the presence of phlobatanins but were absent in both ethanolic and methanolic extracts. Oil and fats were absent in all plant parts, no chemical was used in detection of oil and fats. For reducing sugars distilled water and Fehling solution A and B were used which showed that reducing sugar was present only in the ethanolic extract of the leaves. Whereas, only in methanolic extracts of leaves and flowers reducing sugars were found. Saponin was found only in methanolic extract of fruits whereas, the ethanolic extracts of stem and flowers showed the presence of saponins. Overall results indicated that L. camara contain important metabolites i.e. glycosides, saponins, tannins, flavonoids, phenolic compounds, quinones, cumarines, reducing sugars, phlobatanins, terpenoids and steroids due to which should be used for various infectious diseases, and in numerous traditional medicinal preparations. Furthermore, as the plant is an invasive species and cause harder for native plants so can be utilized for its medicinal purposes in large numbers and can be managed.


Introduction
All the flora are very useful in term of medicines [1, 2] because herbal medicines are used to treat many infectious diseases. These plants possess some important phytochemical compounds such as alkaloids, flavonoids, saponins, phenols occur in the plant root, stem, Leaves, flowers and seeds.
Nowadays natural herbs attain high position due to the presence of many affective reagents which are used for the dermatological infections. Various medicines are produced from secondary metabolites which are used against various disorders [3][4][5][6].
L. camara belonging to family Verbinaceae, within the order Lamiales and it includes about 150 species [7]. It was initially presented in Europe in 1636 [8] and was popularly cultivated in the second half of the nineteenth century [9]. Lantana camara is a wild medicinal plant and is distributive from north to South America. This is now spread throughout 60 countries in Mexico, Florida and Brazil. It is an ornamental flowering plant and grows about 1-3 m in altitude but invasive species of Pakistan. Their leaves are shady green ovate, opposite and are 3-8 cm in length and 3-6 cm wide spread, stem is hairy shrubby, flower is small, tubular in shape and arranged in the form of groups called umbel. Flowers are different in colour it may be red, orange, yellow, white and pink. Usually flowers change its colour due to its maturity. Stamens are four in two pairs, and ovaries are two celled. Inflorescence is dome shaped and about 20-40 sessile flowers. The roots of this plant are very strong and their stem gives out a large number of new shoots after cutting their branches [10]. This plant is used in medicines as well as ornamentally very important. Its leaves exhibit antimicrobial, antifungal and insecticidal activity. They are also very important as ethnomedicines because it is used in various medicines for a lot of disease such as ulcer, catarrhal infection, malaria, tumor, cancer, ulcer, eczema, fevers, cold, chicken pox, asthma, swelling, measles, tetanus and high blood pressure [11]. The vegetative parts of the plant are used for the action of rheumatism [12], for fast-tracking deep respiration, stimulates the intestinal activities etc. also used for carminative and antiseptic properties [13,14]. The main objectives of the research work were to check the presence or absence of phytochemical constituents in L. camara as it is an invasive species in Pakistan so use in large amount for its medicinal purpose to manage the plant.

Materials and methods For primary metabolites Collection of plant
For metabolites detection in L. camara, the aerial parts of the plant were collected from Women University, Swabi, Khyber Pakhtunkhwa Pakistan. For primary metabolite detection fresh parts, i.e. stem, leaves, flower and fruit of the plant were used. These parts were collected and separately washed well with tape water then with distilled water to remove all the impurities. These parts were left for a while to drain all the water, weighed and then ground to form a well paste [14,15]. For the analysis of secondary metabolites all the collected plant parts were cleaned then placed on newspaper separately and tagged them. These Ariel parts are then dried in shade. Each part was crushed and grind into well powdered [3-6]. The entire laboratory work was carried out in the Department of Botany, Women University Swabi. The following methods were adopted for metabolites determination: Lutein 10 g of fresh parts were crushed and dipped in ethanol for some time and then filtered the solution by using filter paper. This filtrate was then poured into the test tubes and centrifuged for 10 minutes at 10,000 rpm. The upper clear white layer was collected with the help of syringes. This supernatant was stored at 4 °C for 10 minutes then checked the optical density with the help of spectrophotometer at the wavelength of 446 nm, also checked the blank optical density, the blank of lutein is acetone or ethanol [16]. Anthocyanin About 0.3 gram of fresh parts was crushed separately. This paste was dipped into the extraction buffer, (for extraction buffer about 45% methanol and 5% concentrated acetic acid were mixed and then cooled) about 3 ml of extraction buffer was then poured into each ground part and then centrifuged at 12000 rpm for 5 minutes, then collected the supernatant and was poured it into new falcon tubes and again centrifuged for 5 minutes at 12000 rpm. The top aqueous layer was collected and checked the optical density about the wavelength is 530 nm/ 657 nm. Blank of anthocyanin was the extraction buffer [17]. Beta carotein For the beta carotein screening, about 95% ethanol was used; 10 g sample was taken in 5 ml of ethanol. Then it was put into water bath for about 20 minutes at 70-80 °C. Then relaxed it for about 5 minutes and collected the supernatant through syringes and was cooled. 15 ml of ethanol was added into the previous ethanol and then cooled for 10 minutes. Taken separating funnel, the supernatant was poured with 5 ml of petroleum ether and 5 ml of cold ethanol and shake for 2 seconds. The dense layer was removed whereas the white upper clear layer was collected and checked the optical density of each sample. Also checked the blank optical density at the wavelength of 436 nm, the blank of beta carotein was petroleum ether [18]. Chlorophyll About 0.2 g of leaves was taken in ethanol at about 80% and then placed it into the water bath for about 1 hour. The supernatant was collected and checked their optical density at about 645 nm and 663 nm [19].

For secondary metabolites Extraction
The dried parts of L. camara were broken into small pieces and ground in a grinder to form well powder. 20 g of plant materials were socked in 90% methanol and ethanol for 14 days and shacked it every day to form a good solution. The crude methanol and ethanol extracts were filtered. The solvent was recollected and the concentrated extract was dried through water bath at 55 o C [6, 20].

Phytochemical analysis
The tests were performed on the Methanolic and ethanolic extracts of L. camara using standard procedures to identify the ingredients [6, 20]. Oil and fats 0.5 ml of the extract was evaporated on a filter paper. The filter paper was then dried and observed for transparency or oily appearance on the filter paper, indicating the presence of fixed oils and fats [16]. Tannins

Results and discussion
By performing tests of primary metabolites such as lutein, beta-carotene and anthracyanines. Different optical density was observed through spectrophotometer as shown in the (Table 1). Results were obtained by the phytochemical analysis of secondary metabolites in different parts of L. camara, L. as shown in (Table 2 & 3). About 15 phytochemicals were screened; only ten phytochemicals were detected in various solvent extracts of the plant. Similar results were also reported by Sushama Raj [25]. These extracts were consisting of free amino acids, proteins, carbohydrates, alkaloids, flavonoids, terpenoids, saponins, phenol and vitamin C. The presence of quinones was observed in the plant, but was found in negligible amount in ethanolic extracts of leaf, flower and fruit but in methanolic extracts only leaf having a negligible amount of cumarines. Phlobatanin was not observed in all parts of L. camara in ethanolic as well as methanolic extracts including leaves, flower, stem and fruit. Furthermore, glycoside was also not observed in ethanolic and methanolic extracts of stem, flower and fruit. Tiwari et al. [26] showed that those factors which affects the solvent are, the quantity of phytochemicals to be extracted, rate of extraction, diversity of different extracted compounds, diversity of inhibitory compounds extracted, affluence of subsequent handling of extracts, toxicity of solvent in a bioassay process and potential health hazard of extract. The results showed that the alkaloids in different plant parts were present in petroleum ether extracts but these were absent in all other plant parts extracts. Proteins, steroids and carbohydrates were present only in alcohol and chloroform. This corroborates the report of Tiwari et al. [27]. The extracts of L. camara were showed that the presence of tannins, flavonoids, terpenoids, saponins, cumarines, quinones, reducing sugars, phenolic compounds and steroids.
These were the major phytochemical groups as shown in (